Chimeric alphavirus replicon particles

ABSTRACT

Chimeric alphaviruses and alphavirus replicon particles are provided including methods of making and using same. Specifically, alphavirus particles are provided having nucleic acid molecules derived from one or more alphaviruses and structural proteins (capsid and/or envelope) from at least two or more alphaviruses. Methods of making, using, and therapeutic preparations containing the chimeric alphavirus particle, are disclosed.

This application claims the benefit of U.S. Ser. No. 60/295,451 filedMay 31, 2001, which application is hereby incorporated by reference inits entirety.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by NIH HIVDDT GrantNo. N01-AI-05396 from the National Institutes of Health. The Governmentmay have certain rights in the invention.

TECHNICAL FIELD

The present invention relates generally to chimeric alphavirusparticles. More specifically, the present invention relates to thepreparation of chimeric alphaviruses having RNA derived from at leastone alphavirus and one or more structural elements (capsid and/orenvelope) derived from at least two different alphaviruses. The chimericalphaviruses of the present invention are useful in the ex vivo and invivo administration of heterologous genes and also have therapeutic orprophylactic applications.

BACKGROUND

Alphaviruses comprise a set of genetically, structurally, andserologically related arthropod-borne viruses of the Togaviridae family.Twenty-six known viruses and virus subtypes have been classified withinthe alphavirus genus, including, Sindbis virus, Semliki Forest virus,Ross River virus, and Venezuelan equine encephalitis virus.

Sindbis virus is the prototype member of the Alphavirus genus of theTogaviridae family. Its replication strategy has been well characterizedin a variety of cultured cells and serves as a model for otheralphaviruses. Briefly, the genome of Sindbis (like other alphaviruses)is an approximately 12 kb single-stranded positive-sense RNA moleculewhich is capped, polyadenylated, and contained within a virus-encodedcapsid protein shell. The nucleocapsid is further surrounded by ahost-derived lipid envelope, into which two viral-specificglycoproteins, E1 and E2, are inserted and anchored by a cytoplasmictail to the nucleocapsid. Certain alphaviruses (e.g., SFV) also maintainan additional protein, E3, which is a cleavage product of the E2precursor protein, PE2.

After virus particle adsorption to target cells, penetration, anduncoating of the nucleocapsid to release viral genomic RNA into thecytoplasm, the replicative process occurs via four alphaviralnonstructural proteins (nsPs), translated from the 5′ two-thirds of theviral genome. Synthesis of a full-length negative strand RNA, in turn,provides template for the synthesis of additional positive strandgenomic RNA and an abundantly expressed 26S subgenomic RNA, initiatedinternally at the junction region promoter. The alphavirus structuralproteins are translated from the subgenomic 26S RNA, which representsthe 3′ one-third of the genome, and like the nsPs, are processedpost-translationally into the individual proteins.

Several members of the alphavirus genus are being developed as“replicon” expression vectors for use as vaccines and therapeutics.Replicon vectors may be utilized in several formats, including DNA, RNA,and recombinant replicon particles. Such replicon vectors have beenderived from alphaviruses that include, for example, Sindbis virus(Xiong et al. (1989) Science 243:1188-1191; Dubensky et al., (1996) J.Virol. 70:508-519; Hariharan et al. (1998) J. Virol. 72:950-958; Polo etal. (1999) PNAS 96:4598-4603), Semliki Forest virus (Liljestrom (1991)Bio/Technology 9:1356-1361; Berglund et al. (1998) Nat. Biotech.16:562-565), and Venezuelan equine encephalitis virus (Pushko et al.(1997) Virology 239:389-401). A wide body of literature has nowdemonstrated efficacy of alphavirus replicon vectors for applicationssuch as vaccines (see for example, Dubensky et al., ibid; Berglund etal., ibid; Hariharan et al., ibid, Pushko et al., ibid; Polo et al.,ibid; Davis et al. (2000) J Virol. 74:371-378; Schlesinger and Dubensky(1999) Curr Opin. Biotechnol. 10:434-439; Berglund et al. (1999) Vaccine17:497-507). Generally, speaking, a “replicon” particle refers to avirus particle containing a self-replicating nucleic acid. The repliconparticle itself is generally considered replication incompetent or“defective,” that is no progeny replicon particles will result when acell is infected with a replicon particle. Through the years, severalsynonymous terms have emerged that are used to describe repliconparticles. These terms include recombinant viral particle, recombinantalphavirus particle, alphavirus replicon particle and replicon particle.However, as used herein, these terms all refer to a virion-like unitcontaining a virus-derived RNA vector replicon, specifically, analphavirus RNA vector replicon. Moreover, these terms may be referred tocollectively as vectors, vector constructs or gene delivery vectors.

Currently, several alphaviruses are being developed as gene deliverysystems for vaccine and other therapeutic applications. Althoughgenerally quite similar in overall characteristics (e.g., structure,replication), individual alphaviruses may exhibit some particularproperty (e.g., receptor binding, interferon sensitivity, and diseaseprofile) that is unique. To exploit the most desirable properties fromeach virus a chimeric replicon particle approach has been developed.Specifically, a chimeric alphavirus replicon particle may have RNAderived from one virus and one or more structural components derivedfrom a different virus. The viral components are generally derived fromclosely related viruses; however, chimeric virus particles made fromdivergent virus families are possible.

It was previously demonstrated that chimeric alphavirus repliconparticles can be generated, wherein the RNA vector is derived from afirst alphavirus and the structural “coat” proteins (e.g., envelopeglycoproteins) are derived from a second alphavirus (see, for exampleU.S. patent application Ser. No. 09/236,140; see also, U.S. Pat. Nos.5,789,245, 5,842,723, and 6,015,694; as well as WO 95/07994, WO 97/38087and WO 99/18226). However, although previously-described strategies weresuccessful for making several alphavirus chimeras, such chimericparticles are not always produced in commercially viable yields, perhapsdue to less efficient interactions between the viral RNA and structuralproteins, resulting in decreased productivity.

Thus, there remains a need for compositions comprising and methods ofmaking and using chimeric replicon particles and replicons, for examplefor use as gene delivery vehicles having altered cell and tissue tropismand/or structural protein surface antigenicity.

SUMMARY

The present invention includes compositions comprising chimericalphaviruses and alphavirus replicon particles and methods of making andusing these particles.

In one aspect, the present invention provides chimeric alphavirusparticles, comprising RNA derived from one or more alphaviruses; andstructural proteins wherein at least one of said structural proteins isderived from two or more alphaviruses. In certain embodiments, the RNAis derived from a first alphavirus and the structural proteins comprise(a) a hybrid capsid protein having (i) an RNA binding domain derivedfrom said first alphavirus and (ii) an envelope glycoprotein interactiondomain derived from a second alphavirus; and (b) an envelopeglycoprotein from said second alphavirus. In other embodiments, the RNAis derived from a first alphavirus and the structural proteins comprise(a) a capsid protein derived from first alphavirus; and (b) an envelopeglycoprotein having (i) a cytoplasmic tail portion and (ii) a remainingportion, wherein the cytoplasmic tail portion is derived from said firstalphavirus and the remaining portion derived from a second alphavirus.The nucleic acid can be derived from a first virus that is containedwithin a viral capsid derived from the same virus but having envelopeglycoprotein components from a second virus. In still furtherembodiments, the chimeric particles comprise hybrid capsid proteins andhybrid envelope proteins. Furthermore, the hybrid proteins typicallycontain at least one functional domain derived from a first alphaviruswhile the remaining portion of the protein is derived from one or moreadditional alphaviruses (e.g., envelope glycoprotein components derivedfrom the first virus, the second virus or a combination of two or moreviruses). The remaining portion can include 25% to 100% (or any valuetherebetween) of sequences derived from different alphaviruses.

Thus, the modified (or chimeric) alphavirus replicon particles of thepresent invention include, but are not limited to, replicon particlescomposed of a nucleic acid derived from one or more alphaviruses(provided by the replicon vector) that is contained within at least onestructural element (capsid and/or envelope protein) derived from two ormore alphaviruses (e.g., provided by defective helpers or otherstructural protein gene expression cassettes). For example, the chimericparticles comprise RNA from a first alphavirus, a hybrid capsid proteinwith an RNA binding domain from the first alphavirus and an envelopeglycoprotein interaction domain from a second alphavirus, and anenvelope glycoprotein from the second alphavirus. In other embodiments,the particles of the present invention comprise RNA from a firstalphavirus, a capsid protein the first alphavirus and an envelopeglycoprotein that has a cytoplasmic tail from the first alphavirus withthe remaining portion of the envelope glycoprotein derived from a secondalphavirus. In still another embodiment, the chimeric alphavirusparticles comprise RNA from a first alphavirus, the RNA having apackaging signal derived from a second alphavirus inserted, for example,in a nonstructural protein gene region that is deleted, and a capsidprotein and envelope glycoprotein from the second alphavirus.

In another aspect, the invention includes chimeric alphavirus particlescomprising (a) RNA encoding one or more nonstructural proteins derivedfrom a first alphavirus and a packaging signal derived from a secondalphavirus different from said first alphavirus (e.g., a packagingsignal inserted into a site selected from the group consisting of thejunction of nsP3 with nsP4, following the open reading frame of nsP4,and a deletion in a nonstructural protein gene); (b) a capsid proteinderived from said second alphavirus; and c) an envelope protein derivedfrom an alphavirus different from said first alphavirus. In certainembodiments, the envelope protein is derived from the second alphavirus.

In any of the chimeric particles described herein, the RNA cancomprises, in 5′ to 3′ order (i) a 5′ sequence required fornonstructural protein-mediated amplification, (ii) a nucleotide sequenceencoding alphavirus nonstructural proteins, (iii) a means for expressinga heterologous nucleic acid (e.g., a viral junction region promoter),(iv) the heterologous nucleic acid sequence (e.g., an immunogen), (v) a3′ sequence required for nonstructural protein-mediated amplification,and (vi) a polyadenylate tract. In certain embodiments, the heterologousnucleic acid sequence replaces an alphavirus structural protein gene.Further, in any of the embodiments described herein, the chimeras arecomprised of sequences derived from Sindbis virus (SIN) and Venezuelanequine encephalitis virus (VEE), for example where the first alphavirusis VEE and the second alphavirus is SIN or where the first alphavirus isVEE and second is SIN.

In other aspects, the invention includes an alphavirus replicon RNAcomprising a 5′ sequence required for nonstructural protein-mediatedamplification, sequences encoding biologically active alphavirusnonstructural proteins, an alphavirus subgenomic promoter, anon-alphavirus heterologous sequence, and a 3′ sequence required fornonstructural protein-mediated amplification, wherein the sequenceencoding at least one of said nonstructural proteins is derived from aBiosafety Level 3 (BSL-3) alphavirus and wherein the sequences of saidreplicon RNA exhibit sequence identity to at least one third but no morethan two-thirds of a genome of a BSL-3 alphavirus. In certainembodiments, cDNA copies of these replicons are included as nucleic acidvector sequences in a Eukaryotic Layered Vector Initiation System(ELVIS) vector, for example an ELVIS vector comprising a 5′ promoterwhich is capable of initiating within a eukaryotic cell the synthesis ofRNA from cDNA, and the nucleic acid vector sequence which is capable ofdirecting its own replication and of expressing a heterologous sequence.The BSL-3 alphavirus can be, for example, Venezuelan equine encephalitisvirus (VEE).

In any of the chimeric particles and replicons described herein, the RNAcan further comprise a heterologous nucleic acid sequences, for example,a therapeutic agent or an immunogen (antigen). The heterologous nucleicacid sequence can replace the structural protein coding sequences.Further the heterologous nucleotide sequence can encode, for example, apolypeptide antigen derived from a pathogen (e.g., an infectious agentsuch as a virus, bacteria, fungus or parasite). In preferredembodiments, the antigen is derived from a human immunodeficiency virus(HIV) (e.g. gag, gp120, gp140, gp160 pol, rev, tat, and nef), ahepatitis C virus (HCV) (e.g., C, E1, E2, NS3, NS4 and NS5), aninfluenza virus (e.g., HA, NA, NP, M), a paramyxovirus such asparainfluenza virus or respiratory syncytial virus or measles virus(e.g., NP, M, F, HN, H), a herpes virus (e.g., glycoprotein B,glycoprotein D), a Filovirus such as Marburg or Ebola virus (e.g., NP,GP), a bunyavirus such as Hantaan virus or Rift Valley fever virus(e.g., G1, G2, N), or a flavivirus such as tick-borne encephalitis virusor West Nile virus (e.g., C, prM, E, NS 1, NS3, NS5). In any ofcompositions or methods described herein, the RNA can further comprise apackaging signal from a second alphavirus inserted within a deletednon-essential region of a nonstructural protein 3 gene (nsP3 gene).

In another aspect, methods of preparing (producing) alphaviral repliconparticles are provided. In certain embodiments, the particles areprepared by introducing any of the replicon and defective helper RNAsdescribed herein into a suitable host cell under conditions that permitformation of the particles. In any of the methods described herein, thedefective helper RNAs can include chimeric and/or hybrid structuralproteins (or sequences encoding these chimeric/hybrid proteins) asdescribed herein. For example, in certain embodiments, the methodcomprises introducing into a host cell: (a) an alphavirus replicon RNAderived from one or more alphaviruses, further containing one or moreheterologous sequence(s); and (b) at least one separate defective helperRNA(s) encoding structural protein(s) absent from the replicon RNA,wherein at least one of said structural proteins is derived from two ormore alphaviruses, wherein alphavirus replicon particles are produced.The replicon RNA can be derived from one or more alphaviruses and thestructural proteins can include one or more hybrid proteins, forexample, a hybrid capsid protein having an RNA binding domain derivedfrom a first alphavirus and an envelope glycoprotein interaction domainderived from a second alphavirus; and/or a hybrid envelope proteinhaving a cytoplasmic tail portion and a remaining portion, wherein thecytoplasmic tail portion is derived from a first alphavirus and theremaining portion of said envelope glycoprotein derived from one or morealphaviruses different than the first.

In yet another aspect, the invention provides a method for producingalphavirus replicon particles, comprising introducing into a host cell(a) an alphavirus replicon RNA encoding one or more nonstructuralproteins from a first alphavirus, a packaging signal derived from asecond alphavirus, (e.g., inserted into a site selected from the groupconsisting of the junction of nsP3 with nsP4, following the nsP4 openreading frame and and a nonstructural protein gene deletion) and one ormore heterologous sequence(s); and (b) at least one separate defectivehelper RNA(s) encoding structural protein(s) absent from the repliconRNA, wherein at least one of said structural proteins is a capsidprotein derived from said second alphavirus, and at least one of saidstructural proteins is an envelope protein derived from an alphavirusdifferent from said first alphavirus.

In yet another aspect, the invention includes alphavirus packaging celllines comprising one or more structural protein expression cassettescomprising sequences encoding one or more structural proteins, whereinat least one of said structural proteins is derived from two or morealphaviruses. In certain embodiments, one or more structural proteinexpression cassettes comprise cDNA copies of a defective helper RNA and,optionally, an alphavirus subgenomic promoter. Further, in any of theseembodiments, the defective helper RNA can direct expression of thestructural protein(s).

In yet another aspect, methods of producing viral replicon particlesusing packaging cell lines are provided. Typically, the methods compriseintroducing, into any of the alphavirus packaging cell lines describedherein, any of the alphavirus replicon RNAs described herein, wherein analphavirus particle comprising one or more heterologous RNA sequence(s)is produced. Thus, in certain embodiments, the RNA will include apackaging signal insertion derived from a different alphavirus. In otherembodiments, the packaging cell comprises three separate RNA molecules,for example, a first defective helper RNA molecule encodes for viralcapsid structural protein(s), a second defective helper RNA moleculeencodes for one or more viral envelope structural glycoprotein(s) and athird replicon RNA vector which comprises genes encoding for requirednonstructural replicase proteins and a heterologous gene of interestsubstituted for viral structural proteins, wherein at least one of theRNA molecules includes sequences derived from two or more alphaviruses.Modifications can be made to any one or more of the separate nucleicacid molecules introduced into the cell (e.g., packaging cell) for thepurpose of generating chimeric alphavirus replicon particles. Forexample, a first defective helper RNA can be prepared having a gene thatencodes for a hybrid capsid protein as described herein. In oneembodiment, the hybrid capsid protein has an RNA binding domain derivedfrom a first alphavirus and a glycoprotein interaction domain from asecond alphavirus. A second defective helper RNA may have a gene orgenes that encodes for an envelope glycoprotein(s) from a secondalphavirus, while the replicon vector RNA is derived from a firstalphavirus. In other embodiments, an RNA replicon vector construct isderived from a first alphavirus having a packaging signal from a secondalphavirus, inserted for example, in a nonstructural protein gene regionthat is deleted. The first and second defective helper RNAs have genesthat encode for capsid protein or envelope proteins from the secondalphavirus. In other embodiments, a chimeric alphavirus repliconparticle is made using a first defective helper RNA encoding a capsidprotein (derived from a first alphavirus that is the same as thereplicon vector source virus) and a second defective helper RNA having agene that encodes for a hybrid envelope glycoprotein having acytoplasmic tail fragment from the same alphavirus as the capsid proteinof the first helper RNA and a surface-exposed “ectodomain” of theglycoprotein derived from a second alphavirus. The tail fragmentinteracts with the capsid protein and a chimeric replicon particlehaving RNA and a capsid derived from a first virus, and an envelopederived primarily from a second virus results.

In another aspect, the invention provides a method for producingalphavirus replicon particles, comprising introducing into a permissiblecell, (a) any of the alphavirus replicon RNAs described hereincomprising control elements and polypeptide-encoding sequences encoding(i) biologically active alphavirus nonstructural proteins and (ii) aheterologous protein, and (b) one or more defective helper RNA(s)comprising control elements and polypeptide-encoding sequences encodingat least one alphavirus structural protein, wherein the control elementscan comprise, in 5′ to 3′ order, a 5′ sequence required fornonstructural protein-mediated amplification, a means for expressing thepolypeptide-encoding sequences, and a 3′ sequence required fornonstructural protein-mediated amplification, and further wherein one ormore of said RNA replicon control elements are different than saiddefective helper RNA control elements; and incubating said cell undersuitable conditions for a time sufficient to permit production ofreplicon particles. In certain embodiments, the replicon RNA and saiddefective helper RNA(s) further comprise a subgenomic 5′-NTR. In otherembodiments, the subgenomic 5′-NTR of the replicon RNA is different thatthe subgenomic 5′-NTR of the defective helper RNA; the 5′ sequencerequired for nonstructural protein-mediated amplification of thereplicon RNA is different than the 5′ sequence required fornonstructural protein-mediated amplification of the defective helperRNA; the 3′ sequence required for nonstructural protein-mediatedamplification of the replicon RNA is different than the 3′ sequencerequired for nonstructural protein-mediated amplification of thedefective helper RNA; and/or the means for expressing saidpolypeptide-encoding sequences of the replicon RNA is different than themeans for expressing said polypeptide-encoding sequences of thedefective helper RNA.

In still further aspects, methods are provided for stimulating an immuneresponse within a warm-blooded animal, comprising the step ofadministering to a warm-blooded animal a preparation of alphavirusreplicon particles according to the present invention expressing one ormore antigens derived from at least one pathogenic agent. In certainembodiments, the antigen is derived from a tumor cell. In otherembodiments, the antigen is derived from an infectious agent (e.g.,virus, bacteria, fungus or parasite). In preferred embodiments, theantigen is derived from a human immunodeficiency virus (HIV) (e.g. gag,gp120, gp140, gp160 po1, rev, tat, and nef), a hepatitis C virus (HCV)(e.g., C, E1, E2, NS3, NS4 and NS5), an influenza virus (e.g., HA, NA,NP, M), a paramyxovirus such as parainfluenza virus or respiratorysyncytial virus or measles virus (e.g., NP, M, F, HN, H), a herpes virus(e.g., glycoprotein B, glycoprotein D), a Filovirus such as Marburg orEbola virus (e.g., NP, GP), a bunyavirus such as Hantaan virus or RiftValley fever virus (e.g., G1, G2, N), or a flavivirus such as tick-borneencephalitis virus or West Nile virus (e.g., C, prM, E, NS1, NS3, NS5).Any of the methods described herein can further comprise the step ofadministering a lymphokine, chemokine and/or cytokine (e.g. IL-2, IL-10,IL-12, gamma interferon, GM-CSF, M-CSF, SLC, MIP3α, and MIP3β). Thelymphokine, chemokine and/or cytokine can be administered as apolypeptide or can be encoded by a polynucleotide (e.g., on the same ora different replicon that encodes the antigen(s)). Alternatively, areplicon particle of the present invention encoding a lymphokine,chemokine and/or cytokine may be used as a to stimulate an immuneresponse.

Thus, in any of the compositions and methods described herein, sequencesare derived from at least two alphaviruses, for example Venezuelanequine encephalitis virus (VEE) and Sindbis virus (SIN).

In other aspects, methods are provided to produce alphavirus repliconparticles and reduce the probability of generating replication-competentvirus (e.g., wild-type virus) during production of said particles,comprising introducing into a permissible cell an alphavirus repliconRNA and one or more defective helper RNA(s) encoding at least onealphavirus structural protein, and incubating said cell under suitableconditions for a time sufficient to permit production of repliconparticles, wherein said replicon RNA comprises a 5′ sequence requiredfor nonstructural protein-mediated amplification, sequences which, whenexpressed, code for biologically active alphavirus nonstructuralproteins, a means to express one or more heterologous sequences, aheterologous sequence that is a protein-encoding gene, said gene beingthe 3′ proximal gene within the replicon, a 3′ sequence required fornonstructural protein-mediated amplification, a polyadenylate tract, andoptionally a subgenomic 5′-NTR; and wherein said defective helper RNAcomprises a 5′ sequence required for nonstructural protein-mediatedamplification, a means to express one or more alphavirus structuralproteins, a gene encoding an alphavirus structural protein, said genebeing the 3′ proximal gene within the defective helper, a 3′ sequencerequired for nonstructural protein-mediated amplification, apolyadenylate tract, and optionally a subgenomic 5′-NTR; and whereinsaid replicon RNA differs from at least one defective helper RNA in atleast one element selected from the group consisting of a 5′ sequencerequired for nonstructural protein-mediated amplification, a means forexpressing a 3′ proximal gene, a subgenomic 5′ NTR, and a 3′ sequencerequired for nonstructural protein-mediated amplification.

These and other aspects and embodiments of the invention will becomeevident upon reference to the following detailed description, attachedfigures and various references set forth herein that describe in moredetail certain procedures or compositions (e.g., plasmids, sequences,etc.).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts Venezuelan equine encephalitis virus (VEE) gene synthesisfragments and restriction sites used for assembly of a VEE replicon.

FIG. 2 depicts the oligonucleotide-based synthesis of VEE nsP fragment2. (SEQ ID NO 51 and SEQ ID NO 52).

FIG. 3 depicts VEE gene synthesis fragments and restriction sites usedfor assembly of structural protein genes.

FIG. 4 depicts hybrid capsid protein (SEQ ID NOS:53 to 58) for theefficient production of chimeric Sindbis virus (SIN)/VEE alphavirusparticles.

FIG. 5 depicts hybrid E2 glycoprotein (SEQ ID NOS:59 to 64) for theefficient production of chimeric SIN/VEE alphavirus particles.

FIG. 6 depicts VEE replicons with heterologous SIN packaging signal forefficient packaging using SIN structural proteins.

FIG. 7 (SEQ ID NOS:65 & 66) depicts SIN packaging signal insertion atnsP4/truncated junction region promoter (as used in Chimera 1A made inaccordance with the teachings of the present invention).

FIG. 8 (SEQ ID NO:67) depicts SIN packaging signal insertion atnsP4/non-truncated junction region promoter (as used in Chimera 1B madein accordance with the teachings of the present invention).

FIG. 9 (SEQ ID NO:68) depicts SIN/VEE packaging Chimera number 2insertion of SIN packaging signal into a VEE nonstructural protein gene(nsP3) deletion.

FIG. 10 (SEQ ID NOS:69 to 88) depicts SIN/VEE packaging chimera number 3insertion of SIN packaging signal at carboxy-terminus of VEE nsP3.

FIG. 11 (SEQ ID NOS:89 to 92) depicts modification of nsP3/nsP4 terminifor SIN packaging signal.

FIG. 12 is a graph showing immunogenicity of alphavirus repliconparticle chimeras expressing an HIV antigen.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained filly in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Edition, 1989); Handbook of Surface and Colloidal Chemistry (Birdi,K. S. ed., CRC Press, 1997); Short Protocols in Molecular Biology, 4thed. (Ausubel et al. eds., 1999, John Wiley & Sons); Molecular BiologyTechniques: An Intensive Laboratory Course, (Ream et al., eds., 1998,Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed.(Newton & Graham eds., 1997, Springer Verlag); Peters and Dalrymple,Fields Virology (2d ed), Fields et al. (eds.), B.N. Raven Press, NewYork, N.Y.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “a particle”includes a mixture of two or more such particles.

Prior to setting forth the invention definitions of certain terms thatwill be used hereinafter are set forth.

A “nucleic acid” molecule can include, but is not limited to,prokaryotic sequences, eukaryotic mRNA or other RNA, cDNA fromeukaryotic mRNA or other RNA, genomic DNA sequences from eukaryotic(e.g., mammalian) DNA, and even synthetic DNA sequences. The term alsocaptures sequences that include any of the known base analogs of DNA andRNA and includes modifications such as deletions, additions andsubstitutions (generally conservative in nature), to the nativesequence. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental. Modifications ofpolynucleotides may have any number of effects including, for example,facilitating expression of the polypeptide product in a host cell.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a “polypeptide” refers to a protein that includesmodifications, such as deletions, additions and substitutions (generallyconservative in nature), to the native sequence, so long as the proteinmaintains the desired activity. These modifications may be deliberate,as through site-directed mutagenesis, or may be accidental, such asthrough mutations of hosts that produce the proteins or errors due toPCR amplification. Furthermore, modifications may be made that have oneor more of the following effects: reducing toxicity; facilitating cellprocessing (e.g., secretion, antigen presentation, etc.); andfacilitating presentation to B-cells and/or T-cells.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Two or more polynucleotide sequences can be compared by determiningtheir “percent identity.” Two or more amino acid sequences likewise canbe compared by determining their “percent identity.” The percentidentity of two sequences, whether nucleic acid or peptide sequences, isgenerally described as the number of exact matches between two alignedsequences divided by the length of the shorter sequence and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981). This algorithm can be extended touse with peptide sequences using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An implementation of this algorithm for nucleic acid and peptidesequences is provided by the Genetics Computer Group (Madison, Wis.) intheir BestFit utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.). Other equally suitable programs for calculating thepercent identity or similarity between sequences are generally known inthe art.

For example, percent identity of a particular nucleotide sequence to areference sequence can be determined using the homology algorithm ofSmith and Waterman with a default scoring table and a gap penalty of sixnucleotide positions. Another method of establishing percent identity inthe context of the present invention is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages, theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated,the “Match” value reflects “sequence identity”. Other suitable programsfor calculating the percent identity or similarity between sequences aregenerally known in the art, such as the alignment program BLAST, whichcan also be used with default parameters. For example, BLASTN and BLASTPcan be used with the following default parameters: genetic codestandard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found on the.

One of skill in the art can readily determine the proper searchparameters to use for a given sequence in the above programs. Forexample, the search parameters may vary based on the size of thesequence in question. Thus, for example, a representative embodiment ofthe present invention would include an isolated polynucleotide having Xcontiguous nucleotides, wherein (i) the X contiguous nucleotides have atleast about 50% identity to Y contiguous nucleotides derived from any ofthe sequences described herein, (ii) X equals Y, and (iii) X is greaterthan or equal to 6 nucleotides and up to 5000 nucleotides, preferablygreater than or equal to 8 nucleotides and up to 5000 nucleotides, morepreferably 10-12 nucleotides and up to 5000 nucleotides, and even morepreferably 15-20 nucleotides, up to the number of nucleotides present inthe full-length sequences described herein (e.g., see the SequenceListing and claims), including all integer values falling within theabove-described ranges.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” typicallyhybridizes under conditions that allow detection of a target nucleicacid sequence of at least about 10-14 nucleotides in length having atleast approximately 70% sequence identity with the sequence of theselected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.).

The term “derived from” is used to identify the alphaviral source ofmolecule (e.g., polynucleotide, polypeptide). A first polynucleotide is“derived from” second polynucleotide if it has the same or substantiallythe same basepair sequence as a region of the second polynucleotide, itscDNA, complements thereof, or if it displays sequence identity asdescribed above. Thus, an alphavirus sequence or polynucleotide is“derived from” a particular alphavirus (e.g., species) if it has (i) thesame or substantially the same sequence as the alphavirus sequence or(ii) displays sequence identity to polypeptides of that alphavirus asdescribed above.

A first polypeptide is “derived from” a second polypeptide if it is (i)encoded by a first polynucleotide derived from a second polynucleotide,or (ii) displays sequence identity to the second polypeptides asdescribed above. Thus, an alphavirus polypeptide (protein) is “derivedfrom” a particular alphavirus if it is (i) encoded by an open readingframe of a polynucleotide of that alphavirus (alphaviralpolynucleotide), or (ii) displays sequence identity, as described above,to polypeptides of that alphavirus.

Both polynucleotide and polypeptide molecules can be physically derivedfrom the alphavirus or produced recombinantly or synthetically, forexample, based on known sequences.

Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), translation terminationsequences, 5′ sequence required for nonstructural protein-mediatedamplification, 3′ sequence required for nonstructural protein-mediatedamplification, and means to express one or more heterologous sequences(e.g., subgenomic junction region promoter), see e.g., McCaughan et al.(1995) PNAS USA 92:5431-5435; Kochetov et al (1998) FEBS Letts.440:351-355.

“Alphavirus RNA replicon vector”, “RNA replicon vector”, “repliconvector” or “replicon” refers to an RNA molecule that is capable ofdirecting its own amplification or self-replication in vivo, within atarget cell. To direct its own amplification, the RNA molecule shouldencode the polymerase(s) necessary to catalyze RNA amplification (e.g.,alphavirus nonstructural proteins nsP1, nsP2, nsP3, nsP4) and alsocontain cis RNA sequences required for replication which are recognizedand utilized by the encoded polymerase(s). An alphavirus RNA vectorreplicon should contain the following ordered elements: 5′ viral orcellular sequences required for nonstructural protein-mediatedamplification (may also be referred to as 5′ CSE, or 5′ cis replicationsequence, or 5′ viral sequences required in cis for replication, or 5′sequence which is capable of initiating transcription of an alphavirus),sequences which, when expressed, code for biologically active alphavirusnonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4), and 3′ viral orcellular sequences required for nonstructural protein-mediatedamplification (may also be referred as 3′ CSE, or 3′ viral sequencesrequired in cis for replication, or an alphavirus RNA polymeraserecognition sequence). The alphavirus RNA vector replicon also shouldcontain a means to express one or more heterologous sequence(s), such asfor example, an IRES or a viral (e.g., alphaviral) subgenomic promoter(e.g., junction region promoter) which may, in certain embodiments, bemodified in order to increase or reduce viral transcription of thesubgenomic fragment, or to decrease homology with defective helper orstructural protein expression cassettes, and one or more heterologoussequence(s) to be expressed. A replicon can also contain additionalsequences, for example, one or more heterologous sequence(s) encodingone or more polypeptides (e.g., a protein-encoding gene or a 3′ proximalgene) and/or a polyadenylate tract.

“Recombinant Alphavirus Particle”, “Alphavirus replicon particle” and“Replicon particle” refers to a virion-like unit containing analphavirus RNA vector replicon. Generally, the recombinant alphavirusparticle comprises one or more alphavirus structural proteins, a lipidenvelope and an RNA vector replicon. Preferably, the recombinantalphavirus particle contains a nucleocapsid structure that is containedwithin a host cell-derived lipid bilayer, such as a plasma membrane, inwhich one or more alphaviral envelope glycoproteins (e.g., E2, E1) areembedded. The particle may also contain other components (e.g.,targeting elements such as biotin, other viral structural proteins orportions thereof, hybrid envelopes, or other receptor binding ligands),which direct the tropism of the particle from which the alphavirus wasderived. Generally, the interaction between alphavirus RNA andstructural protein(s) necessary to efficiently form a replicon particleor nucleocapsid may be an RNA-protein interaction between a capsidprotein and a packaging signal (or packaging sequence) contained withinthe RNA.

“Alphavirus packaging cell line” refers to a cell which contains one ormore alphavirus structural protein expression cassettes and whichproduces recombinant alphavirus particles (replicon particles) afterintroduction of an alphavirus RNA vector replicon, eukaryotic layeredvector initiation system, or recombinant alphavirus particle. Theparental cell may be of mammalian or non-mammalian origin. Withinpreferred embodiments, the packaging cell line is stably transformedwith the structural protein expression cassette(s).

“Defective helper RNA” refers to an RNA molecule that is capable ofbeing amplified and expressing one or more alphavirus structuralproteins within a eukaryotic cell, when that cell also containsfunctional alphavirus nonstructural “replicase” proteins. The alphavirusnonstructural proteins may be expressed within the cell by an alphavirusRNA replicon vector or other means. To permit amplification andstructural protein expression, mediated by alphavirus nonstructuralproteins, the defective helper RNA molecule should contain 5′-end and3′-end RNA sequences required for amplification, which are recognizedand utilized by the nonstructural proteins, as well as a means toexpress one or more alphavirus structural proteins. Thus, an alphavirusdefective helper RNA should contain the following ordered elements: 5′viral or cellular sequences required for RNA amplification by alphavirusnonstructural proteins (also referred to elsewhere as 5′ CSE, or 5′ cisreplication sequence, or 5′ viral sequences required in cis forreplication, or 5′ sequence which is capable of initiating transcriptionof an alphavirus), a means to express one or more alphavirus structuralproteins, gene sequence(s) which, when expressed, codes for one or morealphavirus structural proteins (e.g., C, E2, E1), 3′ viral or cellularsequences required for amplification by alphavirus nonstructuralproteins (also referred to as 3′ CSE, or 3′ viral sequences required incis for replication, or an alphavirus RNA polymerase recognitionsequence), and a preferably a polyadenylate tract. Generally, thedefective helper RNA should not itself encode or express in theirentirety all four alphavirus nonstructural proteins (nsP1, nsP2, nsP3,nsP4), but may encode or express a subset of these proteins or portionsthereof, or contain sequence(s) derived from one or more nonstructuralprotein genes, but which by the nature of their inclusion in thedefective helper do not express nonstructural protein(s) or portionsthereof. As a means to express alphavirus structural protein(s), thedefective helper RNA may contain a viral (e.g., alphaviral) subgenomicpromoter which may, in certain embodiments, be modified to modulatetranscription of the subgenomic fragment, or to decrease homology withreplicon RNA, or alternatively some other means to effect expression ofthe alphavirus structural protein (e.g., internal ribosome entry site,ribosomal readthrough element). Preferably an alphavirus structuralprotein gene is the 3′ proximal gene within the defective helper. Inaddition, it is also preferable that the defective helper RNA does notcontain sequences that facilitate RNA-protein interactions withalphavirus structural protein(s) and packaging into nucleocapsids,virion-like particles or alphavirus replicon particles. A defectivehelper RNA is one specific embodiment of an alphavirus structuralprotein expression cassette.

“Eukaryotic Layered Vector Initiation System” refers to an assembly thatis capable of directing the expression of a sequence or gene ofinterest. The eukaryotic layered vector initiation system should containa 5′ promoter that is capable of initiating in vivo (i.e. within aeukaryotic cell) the synthesis of RNA from cDNA, and a nucleic acidvector sequence (e.g., viral vector) that is capable of directing itsown replication in a eukaryotic cell and also expressing a heterologoussequence. Preferably, the nucleic acid vector sequence is analphavirus-derived sequence and is comprised of 5′ viral or cellularsequences required for nonstructural protein-mediated amplification(also referred to as 5′ CSE, or 5′ cis replication sequence, or 5′ viralsequences required in cis for replication, or 5′ sequence which iscapable of initiating transcription of an alphavirus), as well assequences which, when expressed, code for biologically active alphavirusnonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4), and 3′ viral orcellular sequences required for nonstructural protein-mediatedamplification (also referred to as 3′ CSE, or 3′ viral sequencesrequired in cis for replication, or an alphavirus RNA polymeraserecognition sequence). In addition, the vector sequence may include ameans to express heterologous sequence(s), such as for example, a viral(e.g., alphaviral) subgenomic promoter (e.g., junction region promoter)which may, in certain embodiments, be modified in order to prevent,increase, or reduce viral transcription of the subgenomic fragment, orto decrease homology with defective helper or structural proteinexpression cassettes, and one or more heterologous sequence(s) to beexpressed. Preferably the heterologous sequence(s) comprises aprotein-encoding gene and said gene is the 3′ proximal gene within thevector sequence. The eukaryotic layered vector initiation system mayalso contain a polyadenylation sequence, splice recognition sequences, acatalytic ribozyme processing sequence, a nuclear export signal, and atranscription termination sequence. In certain embodiments, in vivosynthesis of the vector nucleic acid sequence from cDNA may be regulatedby the use of an inducible promoter or subgenomic expression may beinducible through the use of translational regulators or modifiednonstructural proteins.

As used herein, the terms “chimeric alphavirus particle” and “chimericalphavirus replicon particle” refer to a chimera or chimeric particlesuch as a virus, or virus-like particle, specifically modified orengineered to contain a nucleic acid derived from a alphavirus otherthan the alphavirus from which either the capsid and/or envelopeglycoprotein was derived (e.g., from a different virus). In such aparticle, the nucleic acid derived from an alphavirus is an RNA moleculecomprising one of any number of different lengths, including, but notlimited to genome-length (encoding nonstructural and structuralproteins) and replicon-length (deleted of one or more structuralproteins). For example, and not intended as a limitation, chimericreplicon particles made in accordance with the teachings of the presentinvention include Sindbis virus (SIN) replicon RNA within a capsidhaving a Sindbis virus RNA binding domain and a Venezuelan equineencephalitis virus (VEE) envelope glycoprotein interaction domain,surrounded by a VEE glycoprotein envelope.

The term “3′ Proximal Gene” refers to a nucleotide sequence encoding aprotein, which is contained within a replicon vector, Eukaryotic LayeredVector Initiation System, defective helper RNA or structural proteinexpression cassette, and located within a specific position relative toanother element. The position of this 3′ proximal gene should bedetermined with respect to the 3′ sequence required for nonstructuralprotein-mediated amplification (defined above), wherein the 3′ proximalgene is the protein-encoding sequence 5′ (upstream) of, and immediatelypreceding this element. The 3′ proximal gene generally is a heterologoussequence (e.g., antigen-encoding gene) when referring to a repliconvector or Eukaryotic Layered Vector Initiation System, or alternatively,generally is a structural protein gene (e.g., alphavirus C, E2, E1) whenreferring to a defective helper RNA or structural protein expressioncassette.

The term “5′ viral or cellular sequences required for nonstructuralprotein-mediated amplification” or “5′ sequences required fornonstructural protein-mediated amplification” refers to a functionalelement that provides a recognition site at which the virus orvirus-derived vector synthesizes positive strand RNA. Thus, it is thecomplement of the actual sequence contained within the virus or vector,which corresponds to the 3′ end of the of the minus-strand RNA copy,which is bound by the nonstructural protein replicase complex, andpossibly additional host cell factors, from which transcription of thepositive-strand RNA is initiated. A wide variety of sequences may beutilized for this function. For example, the sequence may include thealphavirus 5′-end nontranslated region (NTR) and other adjacentsequences, such as for example sequences through nucleotides 210, 250,300, 350, 400, or 450. Alternatively, non-alphavirus or other sequencesmay be utilized as this element, while maintaining similar functionalcapacity, for example, in the case of SIN, nucleotides 10-75 for tRNAAsparagine (Schlesinger et al., U.S. Pat. No. 5,091,309). The term isused interchangeably with the terms 5′ CSE, or 5′ viral sequencesrequired in cis for replication, or 5′ sequence that is capable ofinitiating transcription of an alphavirus.

The term “viral subgenomic promoter” refers to a sequence of virusorigin that, together with required viral and cellular polymerase(s) andother factors, permits transcription of an RNA molecule of less thangenome length. For an alphavirus (alphaviral) subgenomic promoter oralphavirus (alphaviral) subgenomic junction region promoter, thissequence is derived generally from the region between the nonstructuraland structural protein open reading frames (ORFs) and normally controlstranscription of the subgenomic mRNA. Typically, the alphavirussubgenomic promoter consists of a core sequence that provides mostpromoter-associated activity, as well as flanking regions (e.g.,extended or native promoter) that further enhance thepromoter-associated activity. For example, in the case of the alphavirusprototype, Sindbis virus, the normal subgenomic junction region promotertypically begins at approximately nucleotide number 7579 and continuesthrough at least nucleotide number 7612 (and possibly beyond). At aminimum, nucleotides 7579 to 7602 are believed to serve as the coresequence necessary for transcription of the subgenomic fragment.

The terms “3′ viral or cellular sequences required for nonstructuralprotein-mediated amplification” or “3 ′ sequences required fornonstructural protein-mediated amplification” are used interchangeablywith the terms 3′ CSE, or 3′ cis replication sequences, or 3′ viralsequences required in cis for replication, or an alphavirus RNApolymerase recognition sequence. This sequence is a functional elementthat provides a recognition site at which the virus or virus-derivedvector begins replication (amplification) by synthesis of the negativeRNA strand. A wide variety of sequences may be utilized for thisfunction. For example, the sequence may include a complete alphavirus3′-end non-translated region (NTR), such as for example, with SIN, whichwould include nucleotides 11,647 to 11,703, or a truncated region of the3′ NTR, which still maintains function as a recognition sequence (e.g.,nucleotides 11,684 to 11,703). Other examples of sequences that may beutilized in this context include, but are not limited to, non-alphavirusor other sequences that maintain a similar functional capacity to permitinitiation of negative strand RNA synthesis (e.g., sequences describedin George et al., (2000) J. Virol. 74:9776-9785).

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune system to make a humoral and/or cellular antigen-specificresponse. The term is used interchangeably with the term “immunogen.”Normally, an epitope will include between about 3-15, generally about5-15 amino acids. A B-cell epitope is normally about 5 amino acids butcan be as small as 3-4 amino acids. A T-cell epitope, such as a CTLepitope, will include at least about 7-9 amino acids, and a helperT-cell epitope at least about 12-20 amino acids. Normally, an epitopewill include between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term “antigen” denotes both subunit antigens, (i.e.,antigens which are separate and discrete from a whole organism withwhich the antigen is associated in nature), as well as, killed,attenuated or inactivated bacteria, viruses, fungi, parasites or othermicrobes as well as tumor antigens, including extracellular domains ofcell surface receptors and intracellular portions that may containT-cell epitopes. Antibodies such as anti-idiotype antibodies, orfragments thereof, and synthetic peptide mimotopes, which can mimic anantigen or antigenic determinant, are also captured under the definitionof antigen as used herein. Similarly, an oligonucleotide orpolynucleotide that expresses an antigen or antigenic determinant invivo, such as in gene therapy and DNA immunization applications, is alsoincluded in the definition of antigen herein.

Epitopes of a given protein can be identified using any number ofepitope mapping techniques, well known in the art. See, e.g., EpitopeMapping Protocols in Methods in Molecular Biology, Vol. 66 (Gleun E.Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linearepitopes may be determined by e.g., concurrently synthesizing largenumbers of peptides on solid supports, the peptides corresponding toportions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al. (1984) Proc. Nat'l Acad Sci. USA 81:3998-4002;Geysen et al. (1986) Molec. Immunol 23:709-715, all incorporated hereinby reference in their entireties.

Similarly, conformational epitopes are readily identified by determiningspatial conformation of amino acids such as by, e.g., x-raycrystallography and nuclear magnetic resonance. See, e.g., EpitopeMapping Protocols, supra.

For purposes of the present invention, antigens can be derived fromtumors and/or any of several known viruses, bacteria, parasites andfungi, as described more fully below. The term also intends any of thevarious tumor antigens or any other antigen to which an immune responseis desired. Furthermore, for purposes of the present invention, an“antigen” refers to a protein that includes modifications, such asdeletions, additions and substitutions (generally conservative innature), to the native sequence, so long as the protein maintains theability to elicit an immunological response, as defined herein. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts that producethe antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, including secretory (IgA) orIgG molecules, while a “cellular immune response” is one mediated byT-lymphocytes and/or other white blood cells. One important aspect ofcellular immunity involves an antigen-specific response by cytolyticT-cells (“CTL”s). CTLs have specificity for peptide antigens that arepresented in association with proteins encoded by the majorhistocompatibility complex (MHC) and expressed on the surfaces of cells.CTLs help induce and promote the destruction of intracellular microbes,or the lysis of cells infected with such microbes. Another aspect ofcellular immunity involves an antigen-specific response by helperT-cells. Helper T-cells act to help stimulate the function, and focusthe activity of, nonspecific effector cells against cells displayingpeptide antigens in association with MHC molecules on their surface. A“cellular immune response” also refers to the production of cytokines,chemokines and other such molecules produced by activated T-cells and/orother white blood cells, including those derived from CD4+ and CD8+T-cells. In addition, a chemokine response may be induced by variouswhite blood or endothelial cells in response to an administered antigen.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol (1994) 24:2369-2376. Recent methods of measuringcell-mediated immune response include measurement of intracellularcytokines or cytokine secretion by T-cell populations (e.g., by ELISPOTtechnique), or by measurement of epitope specific T-cells (e.g., by thetetramer technique)(reviewed by McMichael, A. J., and O'Callaghan, C.A., J. Exp. Med. 187(9):1367-1371, 1998; Mcheyzer-Williams, M. G., etal, Immunol. Rev. 150:5-21, 1996; Lalvani, A., et al., J. Exp. Med.186:859-865, 1997).

Thus, an immunological response as used herein may be one thatstimulates CTLs, and/or the production or activation of helper T-cells.The production of chemokines and/or cytokines may also be stimulated.The antigen of interest may also elicit an antibody-mediated immuneresponse. Hence, an immunological response may include one or more ofthe following effects: the production of antibodies (e.g., IgA or IgG)by B-cells; and/or the activation of suppressor, cytotoxic, or helperT-cells and/or γδT-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection to animmunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

An “immunogenic composition” is a composition that comprises anantigenic molecule (or nucleotide sequence encoding an antigenicmolecule) where administration of the composition to a subject resultsin the development in the subject of a humoral and/or a cellular and/ormucosal immune response to the antigenic molecule of interest. Theimmunogenic composition can be introduced directly into a recipientsubject, such as by injection, inhalation, oral, intranasal or any otherparenteral or mucosal (e.g., intra-rectally or intra-vaginally) route ofadministration.

By “subunit vaccine” is meant a vaccine composition that includes one ormore selected antigens but not all antigens, derived from or homologousto, an antigen from a pathogen of interest such as from a virus,bacterium, parasite or fungus. Such a composition is substantially freeof intact pathogen cells or pathogenic particles, or the lysate of suchcells or particles. Thus, a “subunit vaccine” can be prepared from atleast partially purified (preferably substantially purified) immunogenicpolypeptides from the pathogen, or analogs thereof. The method ofobtaining an antigen included in the subunit vaccine can thus includestandard purification techniques, recombinant production, or syntheticproduction.

1.0. Introduction

Several members of the alphavirus genus are being developed as genedelivery systems for vaccine and other therapeutic applications(Schlesinger and Dubensky, Curr. Opin. Biotechnol., 10:434-9 1999). Thetypical “replicon” configuration of alphavirus vector constructs, asdescribed in more detail above and in U.S. Pat. Nos. 5,789,245,5,843,723, 5,814,482, and 6,015,694, and WO 00/61772, comprises a 5′sequence which initiates transcription of alphavirus RNA, a nucleotidesequence encoding alphavirus nonstructural proteins, a viral subgenomicjunction region promoter which directs the expression of an adjacentheterologous nucleic acid sequence, an RNA polymerase recognitionsequence and preferably a polyadenylate tract. Other terminology todefine the same elements is also known in the art.

Often, for in vivo vaccine and therapeutic applications, the alphavirusRNA replicon vector or replicon RNA is first packaged into a virus-likeparticle, comprising the alphavirus structural proteins (e.g., capsidprotein and envelope glycoproteins). Because of their configuration,vector replicons do not express these alphavirus structural proteinsnecessary for packaging into recombinant alphavirus replicon particles.Thus, to generate replicon particles, the structural proteins must beprovided in trans. Packaging may be accomplished by a variety ofmethods, including transient approaches such as co-transfection of invitro transcribed replicon and defective helper RNA(s) (Liljestrom,Bio/Technology 9:1356-1361, 1991; Bredenbeek et al., J. Virol.67:6439-6446, 1993; Frolov et al., J. Virol. 71:2819-2829, 1997; Pushkoet al., Virology 239:389-401, 1997; U.S. Pat. Nos. 5,789,245 and5,842,723) or plasmid DNA-based replicon and defective helper constructs(Dubensky et al., J. Virol. 70:508-519, 1996), as well as introductionof alphavirus replicons into stable packaging cell lines (PCL) (Polo etal., PNAS 96:4598-4603, 1999; U.S. Pat. Nos. 5,789,245, 5,842,723,6,015,694; WO 9738087 and WO 9918226).

The trans packaging methodologies permit the modification of one or morestructural protein genes (for example, to incorporate sequences ofalphavirus variants such as attenuated mutants U.S. Pat. Nos. 5,789,245,5,842,723, 6,015,694), followed by the subsequent incorporation of themodified structural protein into the final replicon particles. Inaddition, such packaging permits the overall modification of alphavirusreplicon particles by packaging of a vector construct or RNA repliconfrom a first alphavirus using structural proteins from a secondalphavirus different from that of the vector construct (WO 95/07994;Polo et al., 1999, ibid; Gardner et al., J. Virol., 74:11849-11857,2000). This approach provides a mechanism to exploit desirableproperties from multiple alphaviruses in a single replicon particle. Forexample, while all alphaviruses are generally quite similar in theiroverall mechanisms of replication and virion structure, the variousmembers of the alphavirus genus can exhibit some unique differences intheir biological properties in vivo (e.g., tropism for lymphoid cells,interferon sensitivity, disease profile). Furthermore, a number ofalphaviruses are classified as Biosafety Level 3 (BSL-3) organisms,which is an issue for particle production (e.g., manufacturing)facilities and possible human use, while others are classified asBiosafety Level 2 (BL-2). Alphavirus replicon particle chimeras providea mechanism to include particular properties of a BSL-3 level alphavirusin a replicon particle derived from a BL-2 level virus. For example,elements from the BSL-3 lymphotropic Venezuelan equine encephalitisvirus (VEE) may be incorporated into a non-naturally lymphotropic BL-2virus (e.g., Sindbis virus).

However, to date, there has been limited success in efficiently androutinely produce commercially acceptable high titer preparations ofchimeric alphavirus particles. Such chimeric alphavirus particles aredesirable for several reasons including specified tropisms or tissuespecificity, altered surface antigenicity and altered recognition by thehost. In this regard, an animal's immune system generally recognizesviral surface antigens, such as the envelope glycoproteins, and directsspecific cellular and humoral responses against them long beforeinternal viral antigens such as capsid proteins are exposed to theimmune system. Consequently, if a replicon particle recipient haspre-existing antibodies directed against the vector's surface antigens(a sensitized host) the replicon particle may be attacked and destroyedbefore it could deliver its therapeutic payload to the target tissue.Given that many of the most successful replicon particles are derivedfrom naturally occurring, infectious viruses, it is likely that at leastsome potential replicon particle recipients have been previously exposedto, and developed immune responses against, surface antigens that arecommon between the replicon particle and the natural infectious virus.The likelihood of an adverse immune response is also increased uponmultiple administrations. Therefore, in order reduce or eliminate thispossibility, subsequent gene delivery replicon particles can be madeusing chimeric replicon particles so the recipient is not required tosee the same structural proteins multiple times.

Described herein are chimeric alphavirus particles that exhibitefficient structural interactions. Thus, the present invention providescompositions and methods for constructing and obtaining recombinantchimeric alphavirus particles with significantly increased efficienciesof packaging/production, for example using SIN/VEE chimeras.

Advantages of the present invention include, but are not limited to, (i)providing chimeric alphavirus particles at commercially viable levels;(ii) the ability to reduce the likelihood of undesirable eventsoccurring, for example, recombination and/or structural gene packaging;(iii) providing gene delivery vehicles with specific tissue and celltropisms (e.g., antigen delivery to an antigen-presenting cell such as adendritic cell).

The teachings provided herein allow one of skill in the art to constructchimeric alphavirus particles derived from a wide variety of differentalphaviruses, particularly when sequences of such alphaviruses havealready been published. Eukaryotic Layered Vector Initiation Systems(ELVIS) can also be designed using these chimeric compositions. Byoptimizing the levels of packaging as disclosed herein, chimericreplicon particles may be produced for use in various applicationsincluding in vaccine and therapeutic applications.

2.0.0. Alphavirus Replicons and Particles

As noted above, chimeric particles as described herein typically includeone or more polynucleotide sequences (e.g., RNA). When found inparticles, these polynucleotides are surrounded by (and interact with)one or more structural proteins. Non-limiting examples of polynucleotidesequences and structural proteins that can be used in the practice ofthe invention are described herein.

2.1.0. Nucleotide Components

The particles, vectors and replicons described herein typically includea variety of nucleic acid sequences, both coding and non-codingsequences. It will be apparent that the chimeric compositions describedherein generally comprise less than a complete alphavirus genome (e.g.,contain less than all of the coding and/or non-coding sequencescontained in a genome of an alphavirus).

Further, it should be noted that, for the illustration herein of variouselements useful in the present invention, alphavirus sequences from aheterologous virus are considered as being derived from an alphavirusdifferent from the alphavirus that is the source of nonstructuralproteins used in the replicon to be packaged, regardless of whether theelement being utilized is in the replicon or defective helper RNA (e.g.,during particle production, when both are present).

2.1.1. Non-Coding Polynucleotide Components

The chimeric particles and replicons described herein typically containsequences that code for polypeptides (e.g., structural ornon-structural) as well as non-coding sequences, such as controlelements. Non-limiting examples of non-coding sequences include 5′sequences required for nonstructural protein-mediated amplification, ameans for expressing a 3′ proximal gene, subgenomic mRNA 5′-endnontranslated region (subgenomic 5′ NTR), and 3′ sequences required fornonstructural protein-mediated amplification (U.S. Pat. Nos. 5,843,723;6,015,694; 5,814,482; PCT publications WO 97/38087; WO 00/61772). Itwill be apparent from the teachings herein that one, more than one orall of the sequences described herein can be included in the particles,vectors and/or replicons described herein and, in addition, that one ormore of these sequences can be modified or otherwise manipulatedaccording to the teachings herein.

Thus, the polynucleotides described herein typically include a 5′sequence required for nonstructural protein-mediated amplification.Non-limiting examples of suitable 5′ sequences include control elementssuch as native alphavirus 5′-end from homologous virus, nativealphavirus 5′-end from heterologous virus, non-native DI alphavirus5′-end from homologous virus, non-native DI alphavirus 5′-end fromheterologous virus, non-alphavirus derived viral sequence (e.g.,togavirus, plant virus), cellular RNA derived sequence (e.g., tRNAelement) (e.g., Monroe et al., PNAS 80:3279-3283, 1983),mutations/deletions of any of the above sequences to reduce homology(See, e.g., Niesters et al., J. Virol. 64:4162-4168, 1990; Niesters etal., J. Virol. 64:1639-1647, 1990), and/or minimal 5′ sequence inhelpers (to approx. 200, 250, 300, 350, 400 nucleotides).

The polynucleotide sequences also generally include a means forexpressing a 3′ proximal gene (e.g., a heterologous sequence,polypeptide encoding sequence). Non-limiting examples of such meansinclude control elements such as promoters and the like, for example, anative alphavirus subgenomic promoter from homologous virus, a nativealphavirus subgenomic promoter from heterologous virus, a corealphavirus subgenomic promoter (homologous or heterologous), minimalsequences upstream or downstream from core subgenomic promoter,mutations/deletions/additions of core or native subgenomic promoter, anon-alphavirus derived compatible subgenomic promoter (e.g. plantvirus), an internal ribosome entry site (IRES), and/or a ribosomalreadthrough element (e.g., BiP).

Suitable subgenomic mRNA 5′-end nontranslated regions (subgenomic 5′NTR)include, but are not limited to, a native alphavirus subgenomic 5′NTRfrom homologous virus, a native alphavirus subgenomic 5′NTR fromheterologous virus, a non-alphavirus derived viral 5′NTR (e.g., plantvirus), a cellular gene derived 5′NTR (e.g., β-globin), and/or sequencescontaining mutations, deletions, and/or additions to native alphavirussubgenomic 5′NTR.

Non-limiting examples of suitable 3′ sequences required fornonstructural protein-mediated amplification include control elementssuch as a native alphavirus 3′-end from homologous virus, a nativealphavirus 3′-end from heterologous virus, a non-native DI alphavirus3′-end from homologous virus, a non-native DI alphavirus 3′-end fromheterologous virus, a non-alphavirus derived viral sequence (e.g.,togavirus, plant virus), a cellular RNA derived sequence, sequencescontaining mutations, deletions, or additions of above sequences toreduce homology (See, e.g., Kuhn et al. (1990) J. Virol. 64:1465-1476),minimal sequence in helpers to approx. (20, 30, 50, 100, 200nucleotides) and/or sequences from cell-repaired 3′ alphavirus CSE. Apolyadenylation sequence can also be incorporated, for example, within3′-end sequences. (See, e.g., George et al. (2000) J.

Virol. 74:9776-9785).

2.1.2. Coding Sequences

The compositions described herein may also include one or more sequencescoding for various alphavirus polypeptides, for example one or more ofthe non-structural (nsP1, nsP2, nsP3, nsP4) or structural (e.g., caspid,envelope) alphavirus polypeptides.

As described in Strauss et al. (1984), supra, a wild-type SIN genome is11,703 nucleotides in length, exclusive of the 5′ cap and the3′-terminal poly(A) tract. After the 5′-terminal cap there are 59nucleotides of 5′ nontranslated nucleic acid followed by a reading frameof 7539 nucleotides that encodes the nonstructural polypeptides andwhich is open except for a single opal termination codon. Following 48untranslated bases located in the junction region that separates thenonstructural and structural protein coding sequences, there is an openreading frame 3735 nucleotides long that encodes the structuralproteins. Finally, the 3′ untranslated region is 322 nucleotides long.The nonstructural proteins are translated from the genomic RNA as twopolyprotein precursors. The first includes nsP1, nsP2 and nsP3 is 1896amino acids in length and terminates at an opal codon at position 1897.The fourth nonstructural protein, nsP4, is produced when readthrough ofthe opal codon produces a second polyprotein precursor of length 2513amino acids, which is then cleaved post-translationally.

The approximately boundaries that define the nonstructural protein genesfrom the genomes of three representative and commonly used alphaviruses,SIN, SFV and VEE as follows.

SIN¹ SFV² VEE³ nsP1 (approx. nucleotide  60-1679  86-1696  45-1649boundaries) nsP1 (approx. amino acid  1-540  1-537  1-535 boundaries)nsP2 (approx. nucleotide 1680-4100 1697-4090 1650-4031 boundaries) nsP2(approx. amino acid  541-1347  538-1335  536-1329 boundaries) nsP3(approx. nucleotide 4101-5747 4191-5536 4032-5681 boundaries) nsP3(approx. amino acid 1348-1896 1336-1817 1330-1879 boundaries) nsP4(approx. nucleotide 5769-7598 5537-7378 5703-7523 boundaries) ¹Strausset al. (1984) Virology 133: 92-110 ²Takkinen (1986) Nucleic Acids Res.14: 5667-5682 ³Kinney et al. (1989) Virology 170: 19

A wild-type alphavirus genome also includes sequences encodingstructural proteins. In SIN, the structural proteins are translated froma subgenomic message which begins at nucleotide 7598, is 4106nucleotides in length (exclusive of the poly(A) tract), and iscoterminal with the 3′ end of the genomic RNA. Like the non-structuralproteins, the structural proteins are also translated as a polyproteinprecursor that is cleaved to produce a nucleocapsid protein and twointegral membrane glycoproteins as well as two small peptides notpresent in the mature virion. Thus, the replicons, particles and vectorsof the present invention can include sequences derived from one or morecoding sequences of one or more alphaviruses.

In addition to providing for sequences derived from coding regions ofalphaviruses, the present invention also provides for alphavirusreplicon vectors containing sequences encoding modified alphavirusproteins, for example modified non-structural proteins to reduce theirpropensity for inter-strand transfer (e.g., recombination) betweenreplicon and defective helper RNA, or between two defective helper RNAs,during positive-strand RNA synthesis, negative-strand RNA synthesis, orboth. Such modifications may include, but are not limited to nucleotidemutations, deletions, additions, or sequence substitutions, in whole orin part, such as for example using a hybrid nonstructural proteincomprising sequences from one alphavirus and another virus (e.g.,alphavirus, togavirus, plant virus).

Thus, a variety of sequence modifications are contemplated within thepresent invention. For example, in certain embodiments, there are one ormore deletions in sequences encoding nonstructural protein gene(s). Suchdeletions may be in nonstructural protein (nsP) 1, 2, 3, or 4, as wellas combinations of deletions from more than one nsP gene. For example,and not intended by way of limitation, a deletion may encompass at leastthe nucleotide sequences encoding VEE nsP1 amino acid residues 101-120,450-470, 460-480, 470-490, or 480-500, numbered relative to the sequencein Kinney et al., (1989) Virology 170:19-30, as well as smaller regionsincluded within any of the above.

In another embodiment, a deletion may encompass at least the sequencesencoding VEE nsP2 amino acid residues 9-29, 613-633, 650-670, or740-760, as well as smaller regions included within any of the above. Inanother embodiment, a deletion may encompass at least the sequencesencoding VEE nsP3 amino acid residues 340-370, 350-380, 360-390,370-400, 380-410, 390-420, 400-430, 410-440, 420-450, 430-460, 440-470,450-480, 460-490, 470-500, 480-510, 490-520, 500-530, or 488-522, aswell as smaller regions included within any of the above. In anotherembodiment, the deletion may encompass at least the sequences encodingVEE nsP4 amino acid residues 8-28, or 552-570, as well as smallerregions included within any of the above. It should be noted thatalthough the above amino acid ranges are illustrated using VEE as anexample, similar types of deletions may be utilized in otheralphaviruses.

Generally, while amino acid numbering is somewhat different betweenalphaviruses, primarily due to slight differences in polyproteinlengths, alignments amongst or between sequences from differentalphaviruses provides a means to identify similar regions in otheralphaviruses (see representative alignment in Kinney et al. (1989)Virology 170:19-30). Preferably, the nonstructural protein genedeletions of the present invention are confined to a region or stretchof amino acids considered as non-conserved among multiple alphaviruses.In addition, conserved regions also may be subject to deletion.

2.2. Alphavirus Structural Proteins

The structural proteins surrounding (and in some cases, interactingwith) the alphavirus replicon or vector polynucleotide component(s) caninclude both capsid and envelope proteins. In most instances, thepolynucleotide component(s) are surrounded by the capsid protein(s),which form nucleocapsids. In turn, the nucleocapsid protein issurrounded by a lipid envelope containing the envelope protein(s). Itshould be understood although it is preferred to have both capsid andenvelope proteins, both are not required.

Alphavirus capsid proteins and envelope proteins are described generallyin Strauss et al. (1994) Microbiol. Rev., 58:491-562. The capsid proteinis the N-terminal protein of the alphavirus structural polyprotein, andfollowing processing from the polyprotein, interacts with alphavirus RNAand other capsid protein monomers to form nucleocapsid structures.

Alphavirus envelope glycoproteins (e.g., E2, E1) protrude from theenveloped particle as surface “spikes”, which are functionally involvedin receptor binding and entry into the target cell.

One or both of these structural proteins (or regions thereof) mayinclude one or more modifications as compared to wild-type. “Hybrid”structural proteins (e.g., proteins containing sequences derived fromtwo or more alphaviruses) also find use in the practice of the presentinvention. Hybrid proteins can include one or more regions derived fromdifferent alphaviruses. These regions can be contiguous ornon-contiguous. Preferably, a particular region of the structuralprotein (e.g., a functional regions such as the cytoplasmic tail portionof the envelope protein or the RNA binding domain of the capsid protein)is derived from a first alphavirus. Any amount of the “remaining”sequences of the protein (e.g., any sequences outside the designatedregion) can be derived from one or more alphaviruses that are differentthan the first. It is preferred that between about 25% to 100% (or anypercentage value therebetween) of the “remaining” portion be derivedfrom a different alphavirus, more preferably between about 35% and 100%(or any percentage value therebetween), even more preferably betweenabout 50% and 100% (or any percentage value therebetween). The sequencesderived from the one or more different alphaviruses in the hybrid can becontiguous or non-contiguous, in other words, sequences derived from onealphavirus can be separated by sequences derived from one or moredifferent alphaviruses.

2.3. Modified Biosafety Level-3 Alphavirus Replicon

The compositions and methods described herein also allow for themodification of replicon vectors or Eukaryotic Layered Vector InitiationSystems derived from a BSL-3 alphavirus (e.g., VEE), such that they maybe utilized at a lower classification level (e.g., BSL-2 or BSL-1) byreducing the nucleotide sequence derived from the parental BSL-3alphavirus to more than one-third but less than two-thirdsgenome-length.

Thus, chimeric replicon vectors, particles or ELVIS can be used thatinclude an alphavirus replicon RNA sequence comprising a 5′ sequencerequired for nonstructural protein-mediated amplification, sequencesencoding biologically active alphavirus nonstructural proteins, analphavirus subgenomic promoter, a non-alphavirus heterologous sequence,a 3′ sequence required for nonstructural protein-mediated amplification,and optionally a polyadenylate tract, wherein the sequence encoding atleast one of said nonstructural proteins is derived from a BSL-3 virus,but wherein the replicon RNA contains sequences derived from saidBiosafety Level 3 alphavirus that in total comprise less than two-thirdsgenome-length of the parental Biosafety Level 3 alphavirus.

Thus, the replicon sequences as described herein exhibit no more than66.67% sequence identity to a BSL-3 alphavirus across the entiresequence. In other words, there may be many individual regions ofsequence identity as compared to a BSL-3 genome, but the overallhomology or percent identity to the entire genome-length of a BSL-3 isno more than 66.67% and nor less than 33.33%. Preferably, the repliconsequences derived from said Biosafety Level 3 alphavirus comprisebetween 40% and two-thirds genome-length of the parental Biosafety Level3 alphavirus. More preferably, the replicon sequences derived from saidBiosafety Level 3 alphavirus comprise between 50% and two-thirdsgenome-length of the parental Biosafety Level 3 alphavirus. Even morepreferably, the replicon sequences derived from said Biosafety Level 3alphavirus comprise between 55% and two-thirds genome-length of theparental Biosafety Level 3 alphavirus. Most preferably, the repliconsequences derived from said Biosafety Level 3 alphavirus comprisebetween 60% and two-thirds genome-length of the parental Biosafety Level3 alphavirus.

As used herein, the definitions of Biosafety Level (e.g., BiosafetyLevel 2, 3, 4) are considered to be those of HHS Publication “Biosafetyin Microbiological and Biomedical Laboratories”, from the U.S.Department of Health and Human Services (Public Health Service, Centersfor Disease Control and Prevention, National Institutes of Health),excerpts of which pertain to such classifications are incorporatedbelow.

Biosafety Level 1 practices, safety equipment, and facility design andconstruction are appropriate for undergraduate and secondary educationaltraining and teaching laboratories, and for other laboratories in whichwork is done with defined and characterized strains of viablemicroorganisms not known to consistently cause disease in healthy adulthumans. Bacillus subtilis, Naegleria grubri, infectious canine hepatitisvirus, and exempt organisms under the NIH Recombinant DNA Guidelines arerepresentative of microorganisms meeting these criteria. Many agents notordinarily associated with disease processes in humans are, however,opportunistic pathogens and may cause infection in the young, the aged,and immunodeficient or immunosuppressed individuals. Vaccine strainsthat have undergone multiple in vivo passages should not be consideredavirulent simply because they are vaccine strains. Biosafety Level 1represents a basic level of containment that relies on standardmicrobiological practices with no special primary or secondary barriersrecommended, other than a sink for handwashing.

Biosafety Level 2 practices, equipment, and facility design andconstruction are applicable to clinical, diagnostic, teaching, and otherlaboratories in which work is done with the broad spectrum of indigenousmoderate-risk agents that are present in the community and associatedwith human disease of varying severity. With good microbiologicaltechniques, these agents can be used safely in activities conducted onthe open bench, provided the potential for producing splashes oraerosols is low. Hepatitis B virus, HIV, the salmonellae, and Toxoplasmaspp. are representative of microorganisms assigned to this containmentlevel. Biosafety Level 2 is appropriate when work is done with anyhuman-derived blood, body fluids, tissues, or primary human cell lineswhere the presence of an infectious agent may be unknown. (Laboratorypersonnel working with human-derived materials should refer to the OSHABloodborne PathogenStandard 2, for specific required precautions).Primary hazards to personnel working with these agents relate toaccidental percutaneous or mucous membrane exposures, or ingestion ofinfectious materials. Extreme caution should be taken with contaminatedneedles or sharp instruments. Even though organisms routinelymanipulated at Biosafety Level 2 are not known to be transmissible bythe aerosol route, procedures with aerosol or high splash potential thatmay increase the risk of such personnel exposure must be conducted inprimary containment equipment, or in devices such as a BSC or safetycentrifuge cups. Other primary barriers should be used as appropriate,such as splash shields, face protection, gowns, and gloves. Secondarybarriers such as handwashing sinks and waste decontamination facilitiesmust be available to reduce potential environmental contamination.

Biosafety Level 3 practices, safety equipment, and facility design andconstruction are applicable to clinical, diagnostic, teaching, research,or production facilities in which work is done with indigenous or exoticagents with a potential for respiratory transmission, and which maycause serious and potentially lethal infection. Mycobacteriumtuberculosis, St. Louis encephalitis virus, and Coxiella burnetii arerepresentative of the microorganisms assigned to this level. Primaryhazards to personnel working with these agents relate toautoinoculation, ingestion, and exposure to infectious aerosols. AtBiosafety Level 3, more emphasis is placed on primary and secondarybarriers to protect personnel in contiguous areas, the community, andthe environment from exposure to potentially infectious aerosols. Forexample, all laboratory manipulations should be performed in a BSC orother enclosed equipment, such as a gas-tight aerosol generationchamber. Secondary barriers for this level include controlled access tothe laboratory and ventilation requirements that minimize the release ofinfectious aerosols from the laboratory.

Non-limiting examples of BSL-3 alphaviruses that may be used in thepractice of the present invention include Cabassou virus, Kyzylagachvirus, Tonate virus, Babanki virus, Venezuelan equine encephalitis virus(excluding TC-83 vaccine strain), Getah virus, Chikungunya virus,Middelburg virus, Sagiyama virus, Everglades virus, Mayaro virus, andMucambo virus.

Biosafety Level 4 practices, safety equipment, and facility design andconstruction are applicable for work with dangerous and exotic agentsthat pose a high individual risk of life-threatening disease, which maybe transmitted via the aerosol route and for which there is no availablevaccine or therapy. Agents with a close or identical antigenicrelationship to Biosafety Level 4 agents also should be handled at thislevel. When sufficient data are obtained, work with these agents m aycontinue at this level or at a lower level. Viruses such as Marburg orCongo-Crimean hemorrhagic fever are manipulated at Biosafety Level 4.The primary hazards to personnel working with Biosafety Level 4 agentsare respiratory exposure to infectious aerosols, mucous membrane orbroken skin exposure to infectious droplets, and autoinoculation. Allmanipulations of potentially infectious diagnostic materials, isolates,and naturally or experimentally infected animals, pose a high risk ofexposure and infection to laboratory personnel, the community, and theenvironment. The laboratory worker's complete isolation from aerosolizedinfectious materials is accomplished primarily by working in a Class IIIBSC or in a fall-body, air-supplied positive-pressure personnel suit.The Biosafety Level 4 facility itself is generally a separate buildingor completely isolated zone with complex, specialized ventilationrequirements and waste management systems to prevent release of viableagents to the environment.

As utilized within the scope of the present invention, creating areplicon that contains more than one-third but less than two-thirds theoriginal genome-length of sequence from any BSL-3 virus (referred to asthe parental virus) may be accomplished in a variety of ways. Forexample, contiguous or non-contiguous regions of the parental virus canbe deleted. Alternatively, contiguous or non-contiguous regions of theparental virus may be utilized. Alternatively, regions of the parentalvirus can be excised and ligated into a BSL-2 or BSL-1 backbone.

In certain embodiments, the alphavirus 5′ and/or 3′ ends (sequencesrequired for nonstructural protein-mediated amplification) are reducedto the minimal nucleotide sequence required to maintain sufficientfunction in the context of a replicon for expression of heterologoussequences, or alternatively replaced by a non-alphavirus sequencecapable of performing the same function. In other embodiments, one ormore alphavirus nonstructural protein genes may be deleted withinspecific regions not well conserved among alphaviruses (e.g., nsP3non-conserved region) or elsewhere. Alternatively, the alphavirussubgenomic promoter region or subgenomic 5′ NTR region may containdeletions. In still further embodiments, one or more structural proteingenes may be deleted, as well as combinations of any of the above.

3.0. Methods of Producing Chimeric Replicon Particles

The chimeric alphavirus replicon particles according to the presentinvention may be produced using a variety of published methods. Suchmethods include, for example, transient packaging approaches, such asthe co-transfection of in vitro transcribed replicon and defectivehelper RNA(s) (Liljestrom, Bio/Technology 9:1356-1361, 1991; Bredenbeeket al., J. Virol. 67:6439-6446, 1993; Frolov et al., J. Virol71:2819-2829, 1997; Pushko et al., Virology 239:389-401, 1997; U.S. Pat.Nos. 5,789,245 and 5,842,723) or plasmid DNA-based replicon anddefective helper constructs (Dubensky et al., J. Virol. 70:508-519,1996), as well as introduction of alphavirus replicons into stablepackaging cell lines (PCL) (Polo et al., PNAS 96:4598-4603, 1999; U.S.Pat. Nos. 5,789,245, 5,842,723, 6,015,694; WO 97/38087, WO 99/18226, WO00/61772, and WO 00/39318).

In preferred embodiments, stable alphavirus packaging cell lines areutilized for replicon particle production. The PCL may be transfectedwith in vitro transcribed replicon RNA, transfected with plasmidDNA-based replicon (e.g., ELVIS vector), or infected with a seed stockof replicon particles, and then incubated under conditions and for atime sufficient to produce high titer packaged replicon particles in theculture supernatant. In particularly preferred embodiments, PCL areutilized in a two-step process, wherein as a first step, a seed stock ofreplicon particles is produced by transfecting the PCL with a plasmidDNA-based replicon. A much larger stock of replicon particles is thenproduced in the second step, by infecting a fresh culture of the PCLwith the seed stock. This infection may be performed using variousmultiplicities of infection (MOI), including a MOI=0.01, 0.05, 0.1, 0.5,1.0, 3,5, or 10. Preferably infection is performed at a low MOI (e.g.,less than 1). Replicon particles at titers even >10⁸ infectious units(IU)/ml can be harvested over time from PCL infected with the seedstock. In addition, the replicon particles can subsequently be passagedin yet larger cultures of naive PCL by repeated low multiplicityinfection, resulting in commercial scale preparations with the same hightiter. Importantly, by using PCL of the “split” structural geneconfiguration, these replicon particle stocks may be produced free fromdetectable contaminating RCV.

As described above, large-scale production of alphavirus repliconparticles may be performed using a bioreactor. Preferably, thebioreactor is an external component bioreactor, which is an integratedmodular bioreactor system for the mass culture, growth, and processcontrol of substrate attached cells. The attachment and propagation ofcells (e.g., alphavirus packaging cells) occurs in a vessel or chamberwith tissue culture treated surfaces, and the cells are with fresh mediafor increased cell productivity. Monitoring and adjustments areperformed for such parameters as gases, temperature, pH, glucose, etc.,and crude vector is harvested using a perfusion pump. Typically, theindividual components of an External Bioreactor separate externalmodules that are connected (i.e., via tubing). The external componentscan be pumps, reservoirs, oxygenators, culture modules, and othernon-standard parts. A representative example of an External ComponentBioreactor is the CellCube™ system (Corning, Inc).

In addition to using the external component bioreactor described herein,a more traditional Stir Tank Bioreactor may also be used, in certaininstances, for alphavirus replicon particle production. In a Stir TankBioreactor, the alphavirus packaging cells may be unattached to anymatrix (i.e., floating in suspension) or attached to a matrix (e.g.,poly disks, micro- or macro carriers, beads). Alternatively, a HollowFiber Culture System may be used.

Following harvest, crude culture supernatants containing the chimericalphavirus replicon particles may be clarified by passing the harvestthrough a filter (e.g., 0.2 uM, 0.45 uM, 0.65 uM, 0.8 uM pore size).Optionally, the crude supernatants may be subjected to low speedcentrifugation prior to filtration to remove large cell debris. Withinone embodiment, an endonuclease (e.g., Benzonase, Sigma #E8263) is addedto the preparation of alphavirus replicon particles before or after achromatographic purification step to digest exogenous nucleic acid.Further, the preparation may be concentrated prior to purification usingone of any widely known methods (e.g., tangential flow filtration).

Crude or clarified alphavirus replicon particles may be concentrated andpurified by chromatographic techniques (e.g., ion exchangechromatography, size exclusion chromatography, hydrophobic interactionchromatography, affinity chromatography). Two or more such purificationmethods may be performed sequentially. In preferred embodiments, atleast one step of ion exchange chromatography is performed and utilizesa ion exchange resin, such as a tentacle ion exchange resin, and atleast one step of size exclusion chromatography is performed. Briefly,clarified alphavirus replicon particle filtrates may be loaded onto acolumn containing a charged ion exchange matrix or resin (e.g., cationor anion exchange). The matrix or resin may consist of a variety ofsubstances, including but not limited to cross-linked agarose, crosslinked polystyrene, cross linked styrene, hydrophilic polyether resin,acrylic resin, and methacrylate based resin. The ion exchanger componentmay comprise, but is not limited to, a cationic exchanger selected fromthe list consisting of sulphopropyl cation exchanger, a carboxymethylcation exchanger, a sulfonic acid exchanger, a methyl sulfonate cationexchanger, and an SO3-exchanger. In other embodiments, the ion exchangercomponent may comprise, but is not limited to, an anionic exchangerselected from the list consisting of DEAE, TMAE, and DMAE. Mostpreferably, ion exchange chromatography is performed using a tentaclecationic exchanger, wherein the ion exchange resin is amethacrylate-based resin with an SO3-cation exchanger (e.g., Fractogel®EDM SO3-).

The chimeric replicon particles may be bound to the ion exchange resinfollowed by one or more washes with buffer containing a salt (e.g., 250mM or less NaCl). Replicon particles then may be eluted from the columnin purified form using a buffer with increased salt concentration. Inpreferred embodiments, the salt concentration is a least 300 mM, 350 mM,400 mM, 450 mM or 500 mM. Elution may be monitored preferably by aspectrophotometer at 280 nm, but also by replicon titer assay, transferof expression (TOE) assay, or protein gel analysis with subsequentCoomassie staining or Western blotting.

The higher salt elution buffer subsequently may be exchanged for a moredesirable buffer, for example, by dilution in the appropriate aqueoussolution or by passing the particle-containing eluate over a molecularexclusion column. Additionally, the use of a molecular size exclusioncolumn may also provide, in certain instances, further purification. Forexample, in one embodiment Sephacryl S-500 or S-400 (Pharmacia)chromatography may be used as both a buffer exchange as well as tofurther purify the fractions containing the replicon particles elutedfrom an ion exchange column. Using this particular resin, the repliconparticles generally are eluted in the late void volume and showimprovement in the level of purity as some of the contaminants aresmaller in molecular weight and are retained on the column longer.However, alternative resins of different compositions as well as sizeexclusion could also be used that might yield similar or improvedresults. In these strategies, larger-sized resins such as SephacrylS-1000 could be incorporated that would allow the replicon particles toenter into the matrix and thus be retained longer, allowingfractionation.

The methods described herein are unlike widely practiced methods inwhich the defective helper RNAs and the replicon vector contain genesderived from the same virus, thereby allowing the process of repliconparticle assembly to proceed naturally and resulting in a repliconparticle having a replicon packaged within a viral capsid and envelopeprotein(s) derived from the same virus that contributed thenonstructural protein genes. Consequently, in such methods, thepackaging signal (also known as packaging sequences), the RNA bindingdomain, the glycoprotein interaction domain and envelope glycoproteinsare all from the same virus.

In contrast, the methods described herein involve the successful andefficient production of alphavirus replicon particles from sequencesderived from two or more alphaviruses. As described herein, theparticles are produced more efficiently and, additionally, have otheradvantages as well.

Methods are also provided to package alphavirus replicon RNA intoreplicon particles (produce replicon particles) and reduce theprobability of generating replication-competent virus (e.g., wild-typevirus) during packaging, comprising introducing into a permissible cellan alphavirus replicon RNA encoding biologically active alphavirusnonstructural proteins and a heterologous polypeptide, together with oneor more defective helper RNA(s) encoding at least one alphavirusstructural protein, and incubating said cell under suitable conditionsfor a time sufficient to permit production of replicon particles. Inthese embodiments, both the replicon RNA and defective helper RNAinclude control elements, particularly a 5′ sequence required fornonstructural protein-mediated amplification, a means to express thepolypeptide-encoding sequences (the polypeptide-encoding sequence(s) arealso referred to as the 3′ proximal gene), for example a promoter thatdrives expression of (1) the heterologous protein in the replicon and(2) the structural proteins in the defective helper RNA, a 3′ sequencerequired for nonstructural protein-mediated amplification, apolyadenylate tract, and, optionally, a subgenomic 5′-NTR. Further,unlike known methods, one or more of these control elements aredifferent (e.g., the sequence is different) as between the RNA in thereplicon and the RNA in the defective helper. For example, in certainembodiments, the 5′ sequence required for nonstructural protein-mediatedamplification is different as between the replicon and the helper RNA.In other embodiments, the means to express the polypeptide-encodingsequences and/or the 3′ sequence required for nonstructuralprotein-mediated amplification is different as between the replicon andthe helper RNA.

One of skill in the art will readily understand that introduction ofreplicon RNA into permissive cells may be performed by a variety ofmeans, such as for example, transfection or electroporation of RNA(e.g., in vitro transcribed RNA), transcription of RNA within the cellfrom DNA (e.g., eukaryotic layered vector initiation system), ordelivery by viral or virus-like particles (e.g., replicon particles) andintroduction of defective helper RNA into permissive cells may also beperformed by a variety of means, such as for example, transfection orelectroporation of RNA (e.g., in vitro transcribed RNA) or transcriptionof RNA within the cell from DNA (e.g., structural protein expressioncassette).

In addition, modifications to reduce homologous sequences may also bemade at the DNA backbone level, such as for example, in a EukaryoticLayered Vector Initiation System or structural protein expressioncassette used for the derivation of packaging cells. Such modificationsinclude, but are not limited to, alternative eukaryotic promoters,polyadenylation sequences, antibiotic resistance markers, bacterialorigins of replication, and other non-functional backbone sequences.

4.0 Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising any of the alphavirus replicon particles, vectors and/orreplicons described herein in combination with a pharmaceuticallyacceptable carrier, diluent, or recipient. Within certain preferredembodiments, a sufficient amount of formulation buffer is added to thepurified replicon particles to form an aqueous suspension. In preferredembodiments, the formulation buffer comprises a saccharide and abuffering component in water, and may also contain one or more aminoacids or a high molecular weight structural additive. The formulationbuffer is added in sufficient amount to reach a desired finalconcentration of the constituents and to minimally dilute the repliconparticles. The aqueous suspension may then be stored, preferably at −70°C., or immediately dried.

The aqueous suspension can be dried by lyophilization or evaporation atambient temperature. Briefly, lyophilization involves the steps ofcooling the aqueous suspension below the gas transition temperature orbelow the eutectic point temperature of the aqueous suspension, andremoving water from the cooled suspension by sublimation to form alyophilized replicon particle. Within one embodiment, aliquots of theformulated recombinant virus are placed into an Edwards RefrigeratedChamber (3 shelf RC3S unit) attached to a freeze dryer (Supermodulyo12K). A multistep freeze drying procedure as described by Phillips etal. (Cryobiology 18:414, 1981) is used to lyophilize the formulatedreplicon particles, preferably from a temperature of −40° C. to −45° C.The resulting composition contains less than 10% water by weight of thelyophilized replicon particles. Once lyophilized, the replicon particlesare stable and may be stored at −20° C. to 25° C., as discussed in moredetail below. In the evaporative method, water is removed from theaqueous suspension at ambient temperature by evaporation. Within oneembodiment, water is removed by a spray-drying process, wherein theaqueous suspension is delivered into a flow of preheated gas, usuallywhich results in the water rapidly evaporating from droplets of thesuspension. Once dehydrated, the recombinant virus is stable and may bestored at −20° C. to 25° C.

The aqueous solutions used for formulation preferably comprise asaccharide, a buffering component, and water. The solution may alsoinclude one or more amino acids and a high molecular weight structuraladditive. This combination of components acts to preserve the activityof the replicon particles upon freezing and also lyophilization ordrying through evaporation. Although a preferred saccharide is lactose,other saccharides may be used, such as sucrose, mannitol, glucose,trehalose, inositol, fructose, maltose or galactose. A particularlypreferred concentration of lactose is 3%-4% by weight.

The high molecular weight structural additive aids in preventingparticle aggregation during freezing and provides structural support inthe lyophilized or dried state. Within the context of the presentinvention, structural additives are considered to be of “high molecularweight” if they are greater than 5000 M.W. A preferred high molecularweight structural additive is human serum albumin. However, othersubstances may also be used, such as hydroxyethyl-cellulose,hydroxymethyl-cellulose, dextran, cellulose, gelatin, or povidone. Aparticularly preferred concentration of human serum albumin is 0.1% byweight.

The buffering component acts to buffer the solution by maintaining arelatively constant pH. A variety of buffers may be used, depending onthe pH range desired, preferably between 7.0 and 7.8. Suitable buffersinclude phosphate buffer and citrate buffer. In addition, it ispreferable that the aqueous solution contains a neutral salt that isused to adjust the final formulated replicon particles to an appropriateiso-osmotic salt concentration. Suitable neutral salts include sodiumchloride, potassium chloride or magnesium chloride. A preferred salt issodium chloride. The lyophilized or dehydrated replicon particles of thepresent invention may be reconstituted using a variety of substances,but are preferably reconstituted using water. In certain instances,dilute salt solutions that bring the final formulation to isotonicitymay also be used.

5.0 Applications

The chimeric alphavirus particles can be used to deliver a wide varietyof nucleotide sequences including, for example, sequences which encodelymphokines or cytokines (e.g., IL-2, IL-12, GM-CSF), prodrug convertingenzymes (e.g., HSV-TK, VZV-TK), antigens which stimulate an immuneresponse (e.g., HIV, HCV, tumor antigens), therapeutic molecules such asgrowth or regulatory factors (e.g., VEGF, FGF, PDGF, BMP), proteinswhich assist or inhibit an immune response, as well as ribozymes andantisense sequences. The above nucleotide sequences include thosereferenced previously (e.g., U.S. Pat. No. 6,015,686, WO 9738087 and WO9918226), and may be obtained from repositories, readily cloned fromcellular or other RNA using published sequences, or synthesized, forexample, on an Applied Biosystems Inc. DNA synthesizer (e.g., APB DNAsynthesizer model 392 (Foster City, Calif.)).

For purposes of the present invention, virtually any polypeptide orpolynucleotide can be used. Antigens can be derived from any of severalknown viruses, bacteria, parasites and fungi, as well as any of thevarious tumor antigens or any other antigen to which an immune responseis desired. Furthermore, for purposes of the present invention, an“antigen” refers to a protein that includes modifications, such asdeletions, additions and substitutions (generally conservative innature), to the native sequence, so long as the protein maintains theability to elicit an immunological response. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts that produce the antigens.

Antigens may be used alone or in any combination. (See, e.g., WO02/00249 describing the use of combinations of bacterial antigens). Thecombinations may include multiple antigens from the same pathogen,multiple antigens from different pathogens or multiple antigens from thesame and from different pathogens. Thus, bacterial, viral, tumor and/orother antigens may be included in the same composition or may beadministered to the same subject separately. It is generally preferredthat combinations of antigens be used to raise an immune response beused in combinations.

Non-limiting examples of bacterial pathogens include diphtheria (See,e.g., Chapter 3 of Vaccines, 1998, eds. Plotkin & Mortimer (ISBN0-7216-1946-0), staphylococcus (e.g., Staphylococcus aureus as describedin Kuroda et al. (2001) Lancet 357:1225-1240), cholera, tuberculosis, C.tetani, also known as tetanus (See, e.g., Chapter 4 of Vaccines, 1998,eds. Plotkin & Mortimer (ISBN 0-7216-1946-0), Group A and Group Bstreptococcus (including Streptococcus pneumoniae, Streptococcusagalactiae and Streptococcus pyogenes as described, for example, inWatson et al. (2000) Pediatr. Infect. Dis. J. 19:331-332; Rubin et al.(2000) Pediatr Clin. North Am. 47:269-284; Jedrzejas et al. (2001)Microbiol Mol Biol Rev 65:187-207; Schuchat (1999) Lancet 353:51-56; GBpatent applications 0026333.5; 0028727.6; 015640.7; Dale et al. (1999)Infect Dis Clin North Am 13:227-1243; Ferretti et al. (2001) PNAS USA98:4658-4663), pertussis (See, e.g., Gusttafsson et al. (1996) N. Engl.J. Med. 334:349-355; Rappuoli et al. (1991) TIBTECH 9:232-238),meningitis, Moraxella catarrhalis (See, e.g., McMichael (2000) Vaccine19 Suppl. 1:S 101-107) and other pathogenic states, including, withoutlimitation, Neisseria meningitides (A, B, C, Y), Neisseria gonorrhoeae(See, e.g., WO 99/24578; WO 99/36544; and WO 99/57280), Helicobacterpylori (e.g., CagA, VacA, NAP, HopX, HopY and/or urease as described,for example, WO 93/18150; WO 99/53310; WO 98/04702) and Haemophilusinfluenza. Hemophilus influenza type B (HIB) (See, e.g., Costantino etal. (1999) Vaccine 17:1251-1263), Porphyromonas gingivalis (Ross et al.(2001) Vaccine 19:4135-4132) and combinations thereof.

Non-limiting examples of viral pathogens include meningitis, rhinovirus,influenza (Kawaoka et al., Virology (1990) 179:759-767; Webster et al.,“Antigenic variation among type A influenza viruses,” p. 127-168. In: P.Palese and D. W. Kingsbury (ed.), Genetics of influenza viruses.Springer-Verlag, New York), respiratory syncytial virus (RSV),parainfluenza virus (PIV), and the like. Antigens derived from otherviruses will also find use in the present invention, such as withoutlimitation, proteins from members of the families Picomaviridae (e.g.,polioviruses, etc. as described, for example, in Sutter et al. (2000)Pediatr Clin North Am 47:287-308; Zimmerman & Spann (1999) Am FamPhysician 59:113-118; 125-126); Caliciviridae; Togaviridae (e.g.,rubella virus, dengue virus, etc.); the family Flaviviridae, includingthe genera flavivirus (e.g., yellow fever virus, Japanese encephalitisvirus, serotypes of Dengue virus, tick borne encephalitis virus, WestNile virus); pestivirus (e.g., classical porcine fever virus, bovineviral diarrhea virus, border disease virus); and hepacivirus (e.g.,hepatitis A, B and C as described, for example, in U.S. Pat. Nos.4,702,909; 5,011,915; 5,698,390; 6,027,729; and 6,297,048); Parvovirsus(e.g., parvovirus B19); Coronaviridae; Reoviridae; Bimaviridae;Rhabodoviridae (e.g., rabies virus, etc. as described for example inDressen et al. (1997) Vaccine 15 Suppl:s2-6; MMWR Morb Mortal Wkly Rep.1998 Jan. 16:47(1):12, 19); Filoviridae; Paramyxoviridae (e.g., mumpsvirus, measles virus, rubella, respiratory syncytial virus, etc. asdescribed in Chapters 9 to 11 of Vaccines, 1998, eds. Plotkin & Mortimer(ISBN 0-7216-1946-0); Orthomyxoviridae (e.g., influenza virus types A, Band C, etc. as described in Chapter 19 of Vaccines, 1998, eds. Plotkin &Mortimer (ISBN 0-7216-1946-0),.); Bunyaviridae; Arenaviridae;Retroviradae (e.g., HTLV-1; HTLV-11; HIV-1 (also known as HTLV-III, LAV,ARV, HTI,R, etc.)), including but not limited to antigens from theisolates HIVI11b, HIVSF2, HIVLAV, HIVI-AL, I-IIVMN); HIV-I CM235,HIV-IIJS4; HIV-2; simian immunodeficiency virus (SIV) among others.Additionally, antigens may also be derived from human papilloma virus(HPV) and the tick-borne encephalitis viruses. See, e.g. Virology, 3rdEdition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B.N. Fields and D. M. Knipe, eds, 1991), for a description of these andother viruses.

Antigens from the hepatitis family of viruses, including hepatitis Avirus (HAV) (See, e.g., Bell et al. (2000) Pediatr Infect Dis. J.19:1187-1188; Iwarson (1995) APMIS 103:321-326), hepatitis B virus (HBV)(See, e.g., Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80),hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis Evirus (HEV) and hepatitis G virus (HGV), can also be conveniently usedin the techniques described herein. By way of example, the viral genomicsequence of HCV is known, as are methods for obtaining the sequence.See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; andWO 90/14436. Also included in the invention are molecular variants ofsuch polypeptides, for example as described in PCT/US99/31245;PCT/US99/31273 and PCT/US99/31272.

Non-limiting examples of tumor antigens include antigens recognized byCD8+ lymphocytes (e.g., melanoma-melanocyte differentiation antigenssuch as MART-1, gp100, tyrosinase, tyrosinase related protein-1,tyrosinase related protein-2, melanocyte-stimulating hormone receptor;mutated antigens such as beta-catenin, MUM-1, CDK-4, caspase-8, KIA0205, HLA-A2-R1701; cancer-testes antigens such as MAGE-1, MAGE-2,MAGE-3, MAGE-12, BAGE, GAGE and NY-ESO-1; and non-mutated sharedantigens over expressed on cancer such as alpha-fetoprotein, telomerasecatalytic protein, G-250, MUC-1, carcinoembryonic antigen, p53,Her-2-neu) as well as antigens recognized by CD4+ lymphocytes (e.g.,gp100, MAGE-1, MAGE-3, tyrosinase, NY-ESO-1, triosephosphate isomerase,CDC-27, and LDLR-FUT). See, also, WO 91/02062, U.S. Pat. No. 6,015,567,WO 01/08636, WO 96/30514, U.S. Pat. Nos. 5,846,538 and 5,869,445.

In certain embodiments, the tumor antigen(s) may be used. Tumor antigensare derived from mutated or altered cellular components. Afteralteration, the cellular components no longer perform their regulatoryfunctions, and hence the cell may experience uncontrolled growth.Representative examples of altered cellular components include ras, p53,Rb, altered protein encoded by the Wilms' tumor gene, ubiquitin, mucin,protein encoded by the DCC, APC, and MCC genes, as well as receptors orreceptor-like structures such as neu, thyroid hormone receptor, plateletderived growth factor (PDGF) receptor, insulin receptor, epidermalgrowth factor (EGF) receptor, and the colony stimulating factor (CSF)receptor. These as well as other cellular components are described forexample in U.S. Pat. No. 5,693,522 and references cited therein.

The present invention also provides methods for delivering theseselected heterologous sequences to a warm-blooded mammal (e.g., a mammalsuch as a human or other warm-blooded animal such as a horse, cow, pig,sheep, dog, cat, rat or mouse) for use as a vaccine or therapeutic,comprising the step of administering to the mammal replicon particles oreukaryotic layered vector initiation systems as described herein, whichare capable of expressing the selected heterologous sequence. Deliverymay be by a variety of routes (e.g., intravenously, intramuscularly,intradermally, intraperitoneally, subcutaneously, orally, intraocularly,intranasally, rectally, intratumorally). In addition, the repliconparticles may either be administered directly (i.e., in vivo), or tocells that have been removed (ex vivo), and subsequently returned to thewarm-blooded mammal.

It should be noted that the selected method for production of chimericalphavirus replicon particles of the present invention should usetechniques known in the art to minimize the possibility of generatingcontaminating replication-competent virus (RCV). One such strategy isthe use of defective helpers or PCL that contain “split” structuralprotein expression cassettes (see U.S. Pat. Nos. 5,789,245; 6,242,259;6,329,201). In this context, the alphavirus structural protein genes aresegregated into separate expression constructs (e.g., capsid separatefrom glycoproteins) such that recombination to regenerate a completecomplement of structural proteins is highly unlikely. The presentinvention also provides compositions and methods to further reduce theprobability of recombination events during production of alphavirusreplicon particles, beyond those conventional methods known in the art.For example, any of the several functional elements (e.g., controlelements) commonly shared by replicon and defective helper RNA, orshared between multiple defective helper RNAs (also eukaryotic layeredvector initiation systems and structural protein expression cassettes)may be substituted with alternative elements that perform the samefunction. In this instance, homology between RNA molecules is decreasedor eliminated. Alternatively, the likelihood of polymerase templateswitching between RNA molecules also may be reduced. Representativefunctional elements commonly shared by replicon and defective helperRNA, or shared between multiple defective helper RNAs, as well as somealternatives for each as contemplated within the present invention areincluded, but not limited to those described above in Section B above.

The following examples are included to more fully illustrate the presentinvention. Additionally, these examples provide preferred embodiments ofthe invention and are not meant to limit the scope thereof.

EXAMPLES Example 1 Construction of a VEE Derived Replicon Vector

In order to construct VEE derived replicon vectors and defective helperpackaging cassettes for use in producing chimeric particles, it wasnecessary to first synthesize complementary DNA corresponding to theentire VEE genome. Based on previously published sequence from thewild-type Trinidad Donkey strain of VEE (GENBANK, L01442), (hereinafterVEE-TRD) the entire 11,447 genome was synthesized and cloned in multiplefragments using overlapping oligonucleotides. Nonstructural protein geneclones were used for assembly of a replicon vector, while the structuralprotein gene clones were used for assembly of defective helper packagingcassettes.

The sequences encoding VEE-TRD nonstructural protein genes were analyzedfor suitable unique restriction cleavage sites that would subdivide theregion into fragments of practical length and which could beconveniently used for final assembly of the complete replicon vectorconstruct. As shown in FIG. 1, a total of 13 intermediate fragments wereidentified, ranging in length from 334 to 723 nucleotides. This seriesof fragments was synthesized using overlapping oligonucleotides andtechniques commonly employed by those of skill in the art of molecularbiology (see below, for example). To the terminal fragments #1 and #13were appended additional sequences necessary to for final constructionof the plasmid that could be used for transcription of RNA repliconexpression vectors in vitro and in vivo. Upstream, as part of fragment1, was placed either a bacteriophage SP6 promoter or eukaryotic CMVpromoter to allow for 30 transcription of replicon RNA. Downstream, aspart of fragment 13, was replicated the viral 3′ UTR, a synthetic A40polyadenylation tract, and the hepatitis delta virus antigenomicribozyme for generation of authentically terminated RNAs (Dubensky etal., J. Virol. 70:508-519, 1996; Polo, 1999, ibid).

As a detailed example of gene synthesis for one of the fragments, fallduplex DNA strands for replicon fragment #2 were generated fromoverlapping synthetic oligonucleotides as described below and shown inFIG. 2 (adjacent oligonucleotides are shown with or without shading tohighlight junctions). First, the fall duplex strand was appended withthe recognition sequence of convenient restriction enzyme sites suitablefor insertion into intermediate cloning vectors. Each fragment was thensubdivided into a series of oligonucleotides with an average length of60 nucleotides each, and overlapping those oligonucleotides from theopposite strand by an average of 20 nucleotides at either end. Synthesisof the initial oligonucleotides was performed by commercial vendors(e.g., Integrated DNA Technologies, Coralville, Iowa; OperonTechnologies, Alameda, Calif.). Oligonucleotides for each fragment werere-constituted as per the supplier's recommendation to yield 100 nMsolutions of each individual oligo. To assemble the fragment, 100 pmolesof each oligo was mixed in a single reaction tube containing T4polynucleotide kinase buffer (New England Biolabs, Beverly, Mass.), 1 mMrATP, water, and 10 units of T4 polynucleotide kinase enzyme (NewEngland Biolabs, Beverly, Mass.) in a final reaction volume of 500 ul.The phosphorylation reaction was allowed to proceed for 30 minutes at37° C., at which time the reaction was supplemented with an additional10 units of T4 polynucleotide kinase and allowed to continue for anadditional 30 minutes. At the conclusion of the reaction, the tubecontaining the mixture was heated to 95° C. for 5′ in a beakercontaining a large volume of water to denature the enzyme and any DNAstrands that may have already annealed. The beaker was then removed fromthe heat source and allowed to slowly cool to ambient temperature, inorder for the complementary oligonucleotides to anneal into full duplexDNA strands.

Once cooled, 0.2 pmoles of the reacted material was ligated with 100pmoles of previously prepared shuttle vector DNA and transformed intocompetent bacteria according to standard methods. Transformants arisingfrom this ligation were analyzed first for the presence of theappropriate terminal replicon enzyme sites, for insert size, andevidence of insert duplication. Several positive transformants wererandomly chosen and submitted for sequence confirmation. Any detectedsequence errors were corrected by fragment swap between two or moresequenced samples, or by site-directed mutagenesis, and re-confirmed forauthenticity.

After all fragments were obtained, final assembly of a replicon vector,similar to those published previously with a variety of alphaviruses(Xiong et al., Science 243:1188-1191, 1989; Dubensky et al., 1996, ibid;Liljestrom et al., Bio/Technol. 9:1356-1361, 1991; Pushko et al.,Virology 239:389-401), was performed by piecing each sub-fragmenttogether with its adjoining fragment through ligation at the previouslyselected terminal fragment cleavage sites. Once assembled, the sequenceof the entire synthetic VEE replicon was reconfirmed. The resultingVEE-based alphavirus vector construct from which replicon RNA can betranscribed in vitro was designated pVCR.

In addition to the SP6 promoter-based vector replicon construct, aVEE-based eukaryotic layered vector initiation system (ELVIS, see U.S.Pat. Nos. 5,814,482 and 6,015,686), which utilized a CMV promoter forlaunching functional RNA replicons from within a eukaryotic cell, alsowas constructed. Modification of plasmid pVCR for conversion into anELVIS vector was accomplished as follows. An existing Sindbis virus(SIN) based ELVIS vector, pSINCP, was used as a donor source for theappropriate backbone components including the CMV promoter, Kanamycinresistance gene, and origin of replication. This strategy was possiblebecause both pSINCP and pVCR share identical sequence elements (e.g.,synthetic polyA sequence, HDV ribozyme) downstream of the nonstructuralgene and viral 3′ UTR regions. In pVCR, the HDV ribozyme is flanked by aunique PmeI site, while in pSINCP the ribozyme is flanked by a BclIsite. The PmeI/BclI fusion then served as the 5′ joining site betweenpSINCP and pVCR. The 3′ joining site was a fortuitous BspEI site presentin nsP1 of both SIN and VEE. In order to accomplish the backbone swap,pSINCP was first transformed into a Dam/Dcm-minus host bacteria, SCS110(Stratagene, La Jolla, Calif.) to obtain DNA cleavable by BclI. A 1203base pair fragment containing the BGHt on the 5′ end and Kan R gene onthe 3′ end was isolated and blunted by means of T4 DNA polymerase (NewEngland Biolabs, Beverly, Mass.) following standard methods. Thisfragment was subsequently further digested with Pst I to liberate a 999bp BclI-PstI fragment that was purified containing the BGHt and the 5′⅔of the Kan R gene.

Plasmid pSINCP contains 4 BspEI sites. To make fragment identificationmore precise, the plasmid was co-digested with NotI, SalI, and Eco47IIIand the 5173 bp fragment was isolated. This fragment was then furtherdigested with PstI and BspEI and from this a 2730 bp PstI-BspEI fragmentwas purified which contained the 3′ ⅓ of the Kan R gene, plasmid originof replication, the CMV pol II promoter, and 420 bp of the Sindbis nsP1gene.

As a source of the 5′ and 3′ end of the VCR replicon, an earlyintermediate, pVCR-DH (see below) was utilized. pVCR-DH containsfragment 1, fragment 13, and all of the terminal restriction sites ofthe intermediate fragments. As such it contains a portion of the VEE-TRDnsP1 gene including the necessary BspEI site and all of the 3′ featuresdescribed above that were necessary for the swap but lacks the corenonstructural region from the 3′ end of nsPI through the 5′ end of nsP4.pVCR-DH was transformed into SCS110 cells as before and digested withBspEI and PmeI to release a 1302 bp fragment containing nsP1′-nsP4′, 3′UTR, A40 tract, and HDV ribozyme.

A three-way ligation of the BclI(blunt-PstI, and PstI-BspEI fragmentsfrom pSINCP, and the BspEI-PmeI fragment from pVCR-DH was performed. Theresulting intermediate was designated pVCPdhintSP. Plasmid pVCPdhintSPwas digested with SacI (cutting 15 bp before the 3′ end of the CMVpromoter) and BspEI at the junction of the Sindbis and VEE sequences innsP1. The vector fragment of this digest was de-phosphorylated andligated with a 326 bp PCR product from pVCR-DH providing the missing 5′terminus of VEE-TRD nsP1. The 5′ primer,[AAGCAGAGCTCGTTTAGTGAACCGTATGGGCGGCGCATG], (SEQ ID NO 1) juxtaposed the3′ terminal 15 nucleotides of the CMV promoter (up to the transcriptionstart site) to the starting base of the VEE 5′ UTR sequence. The 3′primer had the sequence listed[gccctgcgtccagctcatctcgaTCTGTCCGGATCTTCCGC.] (SEQ ID NO 2). Thisintermediate was termed, pVCPdhintf. To complete the construct,pVCPdhintf was digested with NotI and HpaI and the vector fragment wasde-phosphorylated and ligated to the HpaI-NotI fragment of pVCRproviding the missing core VEE nonstructural sequences missing from thepVCPdhintf intermediate. This final VEE-based ELVIS construct wasdesignated pVCP.

Example 2 Construction of Alphavirus Defective Helper Constructs

Prior to construction of defective helpers (DH) of the present inventionfor use in generating hybrid structural protein elements and chimericalphavirus particles, previous existing SIN based defective helperpackaging cassettes (Polo et al., 1999, ibid; Gardner et al., 2000 ibid)were first modified. To generate these new SIN cassettes, plasmidSINBV-neo (Perri et al., J. Virol. 74:9802-9807, 2000) was digested withApaI, treated with T4 DNA polymerase to blunt the ApaI generated-ends,and then digested with BglII and BamHI. The 4.5 kb fragment, whichcontained the plasmid backbone, the SIN subgenomic promoter, SIN 3′-end,synthetic polyA tract, and the HDV antigenomic ribozyme, was gelpurified with QIAquick gel extraction kit and ligated to a 714 bpfragment containing an SP6 promoter and SIN tRNA 5′-end, obtained fromplasmid 47tRNA BBCrrvdel 13 (Frolov et al., J. Virol., 71:2819-2829,1997) which had been previously digested with SacI, treated with T4 DNApolymerase, digested with BamHI and gel purified. Positive clones wereverified by restriction analysis this construct was used as the basisfor insertion via the XhoI-NotI sites (removes existing Neo insert), ofthe alphavirus glycoprotein and capsid sequences described below. TheSIN defective helper cassette backbone described herein is referred toas tDH.

VCR-DH Construction

A polylinker region was cloned into the vector backbone of SINCR-GFP(Gardner et al., 2000, ibid) as a first step. The polylinker containedthe following restriction sites from 5′ to 3′:ApaI-MluI-HpaI-BglII-Bsu36I-PstI-BsaBI-AvrII-SwaI-AspI-BbvCI-AscI-NotI-PineI.To generate the polylinker, the following oligonucleotides were used:

PL1F5′-cacgcgtactactgttaactcatcaagatctactaggcctaaggcaccacctgcaggtagtagatac-(SEQ ID NO 3) acatcataatacc-3′ PL2F5′-tagggcggcgatttaaatgatttagactacgtcagcagccctcagcggcgcgcccacccagcggcc-(SEQ ID NO 4) gcaggatagttt-3′ PL1R5′-tatgatgtgtatctactacctgcaggtggtgccttaggcctagtagatcttgatgagttaacagtagtacgc-(SEQ ID NO 5) gtgggcc-3′ PL2R5′-aaactatcctgcggccgctgggtgggcgcgccgctgagggctgctgacgtagtctaaatcatttaaatcg-(SEQ ID NO 6) ccgccctaggtat-3′Oligonucleotides PL1F and PL1R, and oligonucleotides PL2F and PL2R weremixed in two separate reactions, phosphorylated, denatured, and slowlyannealed. The two reactions were then mixed and ligated to the 2.8 kbfragment generated from plasmid SINCR-GFP that had been previouslydigested with ApaI and PmeI, and gel purified using QIAquick gelextraction kit. Clones were screened for the correct orientation usingAlwNI and NotI restriction digests. The positive clones were verified byrestriction digest with each single enzyme present in the polylinker.This construct was named VCR-backbone. Next, the VEE 3′-end, togetherwith a polyadenylation tract and the HDV ribozyme were inserted into VCRbackbone. This fragment was generated using the following overlappingsynthetic oligonucleotides.

VEE3′-1F5′-ggccgcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatg-3′ (SEQ IDNO 7) VEE3′-1R 5′-ccaatcgccgcgagttctatgtaagcagcttgccaattgctgctgtatgc-3′(SEQ ID NO 8) VEE3′-2F5′-ccgccttaaaatttttattttattttttcttttcttttccgaatcggattttgtttttaat-3′ (SEQID NO 9) VEE3′-2R5′-attaaaaacaaaatccgattcggaaaagaaaagaaaaaataaaataaaaattttaaggcggcatg-3′(SEQ ID NO 10) VEE3′-3F5′-atttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggtcggcatggcatctccacctcctcgcg-3′(SEQ ID NO 11) VEE3′-3R5′-gaccgcgaggaggtggagatgccatgccgacccttttttttttttttttttttttttttttttttttttttttgaaat-3′(SEQ ID NO 12) VEE3′-4F5′-gtccgacctgggcatccgaaggaggacgcacgtccactcggatggctaagggagagccacgttt-3′(SEQ ID NO 13) VEE3′-4R5′-aaacgtggctctcccttagccatccgagtggacgtgcgtcctccttcggatgcccaggtcg-3′ (SEQID NO 14)

Each pair of forward and reverse oligonucleotides (e.g., VEE1F withVEE1R, VEE2F with VEE2R, etc.) were mixed, phosphorylated, denatured,and slowly annealed. Then the 4 pairs of annealed oligonucleotides weremixed together, ligated to each other, digested with enzymes NotI andPmeI, gel purified using a QIAquick gel extraction kit, and ligated tothe VCR-backbone that had been previously digested with the sameenzymes, gel purified and treated with shrimp alkaline phosphatase.Positive clones for the fragment were verified by sequencing. Thisconstruct was called VCR-3′ drib.

Next, the 5′ end of VEE genome was inserted. This fragment was generatedusing overlapping oligonucleotides to cover the genome region of VEEstrain Trinidad donkey (see GENBANK reference, above) from nucleotide 1to the restriction site HpaI. The primers with VEE nucleotide 1 alsocontained an upstream MluI site followed by the SP6 promoter immediately5′ of VEE nucleotide 1. All oligonucleotides were mixed in one reaction,phosphorylated, denatured, slowly annealed, and ligated. Afterinactivating the ligase, the DNA was digested with the enzymes MluI andHpaI, gel purified using the QIAquick gel extraction kit and ligated toVCR-3′ drib that had been previously digested with the same restrictionenzymes, gel purified and treated with shrimp alkaline phosphatase. Thepositive clones for the insert were verified by sequencing. Thisintermediate construct was called VCR-F1-3′ drib.

Finally, the region of VEE containing the subgenomic promoter was clonedinto VCR-F1-3′ drib. This region (fragment 13, FIG. 1) corresponds tothe sequence between restriction site SwaI and nucleotide 7561 of theVEE Trinidad donkey strain genome. The fragment was generated usingoverlapping oligonucleotides corresponding to the Trinidad Donkey strainsequence, with the exception of the oligonucleotide corresponding to the3′ end of the fragment that was modified to carry an additionalrestriction sites (BbvCI) to allow later insertion of heterologoussequences under the control of the subgenomic promoter. Alloligonucleotides were mixed in one reaction, phosphorylated, denatured,slowly annealed, and ligated. After inactivating the ligase, the DNA wasdigested with the enzymes SwaI and BbvCI, gel purified using QIAquickgel extraction kit and ligated to VCR backbone that had been previouslydigested with the same restriction enzymes, gel purified and treatedwith shrimp alkaline phosphatase. Clones positive for the insert wereverified by sequencing and one clone, VF13-14, was subsequently repairedby deleting a small insertion and reconfirming by sequencing. The clonewas next digested with SwaI-NotI, the 600bp fragment was gel purifiedusing the QIAquick gel extraction kit and ligated to VCR-F1-3′ drib thathad been previously digested with the same restriction enzymes, gelpurified and treated with shrimp alkaline phosphatase. The positiveclones for the insert were verified and the construct was called VCR-DH.

Construction of tDH-Vgly, tDH-VE2-120, and tDH-V_(NTR)-glydl160

The VEE Trinidad donkey strain glycoprotein genes were generated usingoverlapping oligonucleotides that were designed based on the publishedGENBANK sequence. To allow expression from the appropriate vectorpackaging cassettes, an ATG codon, in-frame with the glycoprotein geneopen reading frame, was added immediately preceding the first amino acidof E3, and a XhoI site and NotI site were added respectively at the 5′and 3′ end of the glycoprotein gene sequences. Gene synthesis wasperformed by using overlapping PCR to generate five separate fragmentsspanning the entire glycoprotein sequence (FIG. 3). The fragments wereassembled stepwise into a single fragment in pGEM using the restrictionsites indicated in FIG. 3. A small nucleotide deletion within the KasIsites was corrected by standard site-directed mutagenesis. The finalclone was verified by sequencing and designated pGEM-Vgly. Then theglycoprotein gene sequence was transferred from pGEM into tDH using theXhoI-NotI sites and the final clone was designated tDH-Vgly.

A construct similar to tDH-Vgly that also contains the attenuatingmutation at E2 amino acid 120 present in the TC83 vaccine strain of VEEwas constructed in an analogous way. Plasmid pGEM-Vgly was subjected tostandard site directed mutagenesis and the E2-120 mutation confirmed bysequencing. Then, the VEE E2-120 glycoprotein sequence was transferredfrom pGEM into tDH using the XhoI-NotI sites and the construct wasconfirmed by sequencing and designated tDH-VE2-120.

Plasmid tDH-V_(NTR)-glydl160 is a tDH defective helper construct (seeabove) containing a SIN glycoprotein sequence from the human dendriticcell tropic strain described previously (Gardner et al., ibid), in whichthe SIN derived subgenomic 5′ NTR and the synthetic XhoI site weresubstituted by the following VEE subgenomic 5′ NTR sequence(5′-ACTACGACATAGTCTAGTCCGCCAAG) (SEQ ID NO 53). This sequence wasinserted such that it immediately precedes the glycoprotein ATGinitiation codon. The construct is also known as tDH-V_(UTR)-glydl160 toreflect the interchangeable nomenclature for the subgenomic 5′nontranslated region (NTR), also referred to as untranslated region(UTR).

Construction of VCR-DH-Vgly, VCR-DH-VE2-120, and VCR-DH-Sglydl160

The VEE glycoprotein gene sequence between the ATG and the restrictionsite NcoI was amplified by PCR using the following oligonucleotides.

VGBbvCI 5′-atatatatctcgagcctcagcatgtcactagtgaccaccatgt-3′ (SEQ ID NO 15)VGNcoIR 5′-atatataaattccatggtgatggagtcc-3′ (SEQ ID NO 16)After PCR amplification, the fragment was digested with BbvCI and NcoI,and gel purified using QIAquick gel extraction kit. Separately, the VEEE2-120 glycoprotein region from NcoI to NotI was prepared by digestingpGEM-VE2-120 with these enzymes followed by gel purification. The twofragments were mixed and ligated to VCR-DH that had been previouslydigested with BbvCI and NotI, gel purified, and treated with alkalinephosphatase. Positive clones for the insert were verified by sequencingand designated VCR-DH-VE2-120. To obtain VCR-DH-Vgly the NcoI-XbaIfragment was obtained from pGEM-Vgly and used to substitute the samefragment in VCR-DH-VE2-120.

A SIN glycoprotein with the human DC+ phenotype was obtained from adefective helper plasmid E3ndl160/dlRRV, modified from Gardner et al.,(2000, ibid) (PCT WO 01/81609). Plasmid E3ndl160/dlRRV was digested withXhoI, treated it with Klenow fragment to blunt the ends, then digestedwith NotI. The 3 kb fragment was gel purified using QIAquick gelextraction kit and ligated to VCR-DH that had been previously digestedwith BbvCI, treated with Klenow fragment, digested with NotI, andtreated with alkaline phosphatase. A positive clone for the insert wasdesignated VCR-DH Sglydl160. Similarly, a defective helper constructcontaining the SIN LP strain-derived envelope glycoproteins (Gardner etal, 2000, ibid) was constructed.

Construction of VCR-DH-Vcap, VCR-DH-Scap and tDH-Vcap

The VEE capsid gene was synthesized using overlapping oligonucleotides,also designed based on the published GENBANK sequence of the VEETrinidad donkey strain, with the addition of a XhoI site and a Kozakconsensus sequence adjacent to the capsid ATG, and a NotI site at the3′-end. The oligonucleotides were mixed and used for a 25-cycle PCRamplification reaction. The PCR generated fragment was digested with therestriction sites XhoI and NotI, gel purified and cloned into the vectorpBS-SK+. Positive clones for the insert were verified by sequencing.Finally, the capsid sequence was further modified to insert atermination codon at it's 3′-end by PCR amplification in a 25-cyclereaction with the following oligonucleotides.

TRDCtR 5-atatatatgcggccgcttaccattgctcgcagttctccg-3′ (SEQ ID NO 17)contains stop codon in frame with the last amino acid of capsid TRDCtF5′gagatgtcatcgggcacgcatgtgtggtcggagggaagttattc-3′ (SEQ ID NO 18)The product was purified with QIAquick PCR purification kit, digestedwith XhoI and NotI and ligated to the backbone of tDH vector that hadbeen previously prepared by digestion with XhoI and NotI, gelpurification, and alkaline phosphatase treatment. Positive clones forthe insert were verified by sequencing and the construct was designatedtDH-Vcap.

The same PCR product was also digested with XhoI, treated with T4 DNApolymerase to blunt XhoI site, digested with NotI, gel purified, andligated to VCR-DH that had been previously digested with BbvCI, treatedwith T4 DNA polymerase to blunt BbvCI site, digested with NotI, gelpurified, and treated with alkaline phosphatase. Positive clones for theinsert were verified by sequencing and the construct was designatedVCR-DH-Vcap.

The SIN capsid sequence was obtained from a previously describeddefective helper and the 800 bp fragment was gel purified using QIAquickgel extraction kit and ligated to VCR-DH that had been previouslydigested with BbvCI, treated with Klenow fragment, digested with NotI,and treated with alkaline phosphatase. A positive clone for the insertwas designated VCR-DH-Scap.

Example 3 Generation of Alphavirus Replicon Particle Chimeras withHybrid Capsid Protein

In the case of hybrid capsid protein using elements obtained from bothSIN and VEE, a series of hybrid capsid proteins were constructedcontaining the amino terminal (RNA binding) portion from SIN and thecarboxy terminal (glycoprotein interaction) portion from VEE. Additionalconstructs with the opposite portions also were derived. The site atwhich such portions were fused varied by construct and necessarilyfactored into account the differences in overall length of these twocapsid proteins, with SIN capsid being 264 amino acids and VEE capsidbeing 275 amino acids. Sites of fusion to generate the capsid hybridsare indicated in the table below, as well as in FIG. 4.

Name of capsid chimera NH2-terminus COOH-terminus S113V SIN(1-113)VEE(125-275) S129V SIN(1-129) VEE(141-275) S127V SIN(1-127) VEE(139-275)S116V SIN(1-116) VEE(128-275) S109V SIN(1-109) VEE(121-275) V141SVEE(1-141) SIN(130-264)

Each of the hybrid capsid constructs was generated by PCR amplificationof two overlapping fragments, one coding for the amino-terminus ofcapsid protein from SIN or VEE, and the other coding for thecarboxy-terminus of capsid protein from the opposite virus (VEE or SIN,respectively).

Fragments containing SIN capsid sequences were amplified from adefective helper construct (Gardner et al., 2000, ibid), and fragmentscontaining VEE capsid sequences were amplified from constructVCR-DH-Vcap (above). The following oligonucleotides were used:

Fragment 5′ oligonucleotide 3′ oligonucleotide SIN(1-113) SINNtF S113R5′atatatctcgagccaccatgaatag 5′gggaacgtcttgtcggcctccaactaggattctttaacatg-3′ taagtg-3′ (SEQ ID NO 19) (SEQ ID NO 20) containingthe restriction with nt. 1-10 site XhoI (nt. 7-13), the complementary toVEE Kozak consensus sequence capsid sequence and nt .11-31 for optimalprotein to SIN capsid sequence translation (nt. 14-18), and sequencecomplementary to SIN capsid (nts 19-48) SIN(1-129) SINNtF SINNtR5′gaataacttccctccgaccacacat gcgtgcccgatgacatctc-3′ (SEQ ID NO 21) withnt. 1-24 complementary to VEE capsid sequence and nt .25-44 to SINcapsid sequence SIN(1-127) SINNtF S127R 5′ccacacaagcgtacccgatgacatctccgtcttc-3′ (SEQ ID NO 22) with nt. 1-13 complementary to VEE capsidsequence and nt. 14-34 to SIN capsid sequence SIN(1-116) SINNtF S116R5′catgattgggaacaatctgtcggcc tccaac-3′ (SEQ ID NO 23) with nt. 1-9complementary to VEE capsid sequence and nt. 10-31 to SIN capsidsequence SIN(1-109) SINNtF S109R 5′gtcagactccaacttaagtgccatg cg-3′ (SEQID NO 24) with nt. 1-6 complementary to VEE capsid sequence and nt. 7-27to SIN capsid sequence. SIN(130-264) SINCtF SINCtR5′gggaagataaacggctacgctctg 5′atatatatgcggccgctcaccactctgccatggaaggaaagg-3′ tctgtcccttc-3′ (SEQ ID NO 25) (SEQ ID NO 26)complementary to VEE with the restriction site NotI capsid sequence andnt. 22-40 (nt. 9-16) and nt. 17-39 to SIN capsid sequence complementaryto SIN capsid sequence. VEE(125-275) TRD125F TRDCtR5′gccgacaagacgttcccaatcatgt 5′atatatatgcggccgcttaccattgc tggaag-3′tcgcagttctccg-3′ (SEQ ID NO 27) (SEQ ID NO 28) with nt. 1-9complementary with the restriction site NotI to SIN capsid sequence and(nt. 9-16) and nt. 17-39 nt. 10-31 to VEE capsid complementary to VEEsequence capsid VEE(141-275) TRDCtF TRDCtR 5′gagatgtcatcgggcacgcatgtgtggtcggagggaagttattc-3′ (SEQ ID NO 29) with nt. 1-20 complementary toSIN capsid sequence and nt. 21-44 to VEE capsid sequence VEE(139-275)TRD139F TRDCtR 5′-tcatcgggtacgcttgtgtggtcg-3′ (SEQ ID NO 30) with nt.1-8 complementary to SIN capsid sequence and nt. 9-24 to VEE capsidsequence VEE(128-275) TRD128F TRDCtR 5′gacagattgttcccaatcatgttggaaggg-3′ (SEQ ID NO 31) with nt. 1-11 complementary to SIN capsidsequence and nt. 12-30 to VEE capsid sequence VEE(121-275) TRD121FTRDCtR 5′acttaagttggagtctgacaagacg ttcccaatc-3′ (SEQ ID NO 32) with nt.1-13 complementary to SIN capsid sequence and nts. 14-34 to VEE caspidsequence VEE(1-141 TRDNtF TRDNtR 5′atatatctcgagccaccatgttcccg5′-cctttccttccatggccag ttccagccaatg-3′ agcgtagccgtttatcttccc-3′ (SEQ IDNO 33) (SEQ ID NO 34) with the restriction site XhoI with nt. 1-19 (nt.7-13), the Kozak complementary to SIN consensus sequence for capsidsequence and nt. 20-40 optimal protein translation to VEE capsidsequence (nt. 14-18),and nts. 19-48 complementary to VEE capsid sequence

The oligonucleotides listed above were used at 2 μM concentration with0.1 μg of the appropriate template plasmid DNA in a 30 cycle PCRreaction, with Pfu polymerase as suggested by the supplier and with theaddition of 10% DMSO. The general amplification protocol illustratedbelow.

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 60 0.5 10 72 2

The amplified fragments were purified from agarose gel using QIAquickgel extraction kit, and then an aliquot ( 1/15th) of each fragment wasused as template for a second PCR amplification. The two fragments weremixed as follows and amplified with Vent Polymerase as suggested bysupplier, with the addition of 10% DMSO:

-   SIN(1-129)+VEE(141-275)-   SIN(1-127)+VEE(139-275)-   SIN(1-116)+VEE(128-275)-   SIN(1-113)+VEE(125-275)-   SIN(1-109)+VEE(121-275)-   VEE(1-141)+SIN(130-264)    One PCR amplification cycle was performed under the following    conditions:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 42 1 1 72 3For the SIN NH2-terminus/VEE COOH-terminus fusions, the SINNtF andTRDCtR primers, containing the XhoI and NotI restriction sites, wereadded at 2 μM concentration and the complete PCR amplification wasperformed as follows:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 60 0.5 30 72 2The PCR product was purified using the QIAquick kit, digested with XhoIand NotI, gel purified from agarose gel as described above, and ligatedto plasmid tDH that had also been digested with XhoI and NotI to removethe existing capsid gene insert. Clones containing the newly generatedhybrid inserts were verified by sequencing and the new defective helperconstructs for use in producing chimeric particles were designated tDHS129Vcap, tDHS127Vcap, tDHS116Vcap, tDHS113Vcap, and tDHS109Vcap.

Similarly, for the VEE NH2 terminus/SIN COOH terminus fusions, theTRDNtF and SINCtR primers, containing the XhoI and NotI restrictionsites, were added at 2 μM concentration. The PCR amplification wasperformed using the same conditions as above. This PCR fragment was thendigested with XhoI, blunted, digested with NotI and ligated to plasmidVCR-DH-Vcap that had been digested with BbvCI, blunted and digested withNotI. Clones containing the inserts were verified by sequencing and thenew defective helper construct was designated VCR-DH-S129Vcap.

The capsid chimeras were then tested for their efficiencies of repliconpackaging with the appropriate alphavirus replicon vector andglycoprotein defective helper. Specifically, the chimeras with theSIN-derived NH2-terminus and the VEE-derived COOH-terminus were testedfor their ability to package SIN replicons with VEE glycoproteins. Thiswas accomplished as follow. The plasmid DNA encoding for the chimeras(tDHS129Vcap, tDHS127Vcap, tDHS116Vcap, tDHS113Vcap, and tDHS109Vcap)were linearized with the unique restriction site PmeI and used for invitro transcription as described previously (Polo et al., 1999, ibid).Each transcript was co-transfected by electroporation into BHK cellstogether with helper RNA expressing the VEE glycoproteins and SINreplicon RNA expressing GFP, as described previously (Polo et al. 1999,ibid). Transfected cells were incubated at 34° C. for 24 hr, at whichtime the culture supernatants were collected, clarified bycentrifugation, serially diluted, and used to infect naive BHK-21 cellsfor approximately 14 hr. Enumeration of GFP positive cells allowed forquantitation of input vector particles and the vector particle stock.The data below indicate that the efficiency of packaging for a SIN/VEEchimeric particle can be increased quite dramatically, particularly withthe S113V hybrid capsid protein.

Capsid Glycoprotein Replicon Particle titer S129V VEE SIN 4e⁵ IU/mlS127V VEE SIN 2e⁴ IU/ml S116V VEE SIN 1.6e⁶ IU/ml   S113V VEE SIN 1.1e⁷IU/ml  

Similarly, each chimera transcript was co-transfected by electroporationinto BHK cells together with 1) helper RNA expressing the VEEglycoproteins with the E2-120 attenuating mutation tDHVE2-120 and 2) SINreplicon RNA expressing GFP. Transfected cells were incubated at 34° C.for 24 hr, at which time the culture supernatants were collected,clarified by centrifugation, serially diluted, and used to infect naïveBHK-21 cells for approximately 14 hr. Enumeration of GFP positive cellsallowed for quantitation of input vector particles and titerdetermination for the replicon vector particle stock. The data belowconfirm that the hybrid capsid can dramatically increase the packagingefficiency of the SIN replicon in particles containing the VEEglycoproteins.

Capsid Glycoprotein Replicon Particle titer S129V VE2-120 SIN 1.6e⁷IU/ml S127V VE2-120 SIN 5.1e⁵ IU/ml S116V VE2-120 SIN 4.7e⁷ IU/ml S113VVE2-120 SIN 9.3e⁷ IU/ml S VE2-120 SIN   1e² IU/ml

Similar experiments with the VCR-GFP RNA, cotransfected with RNA helperscoding for the hybrid capsid S129Vcap and the SIN glycoproteins,produced particles with average titers of 1.6e7 IU/ml demonstrating thatthe ability of this hybrid protein to efficiently package VEE-derivedvector RNA.

To further maximize the capsid-RNA and capsid-glycoprotein interactions,an additional construct was made, whereby the S113V hybrid capsidprotein gene was incorporated into the genome of a chimeric alphavirus,comprising the 5′-end, 3′-end, subgenomic promoter and nonstructuralprotein genes of SIN, and the glycoprotein genes from VEE.

To generate such construct, an initial genome-length SIN cDNA clone fromwhich infectious RNA may be transcribed in vitro was generated byassembling replicon and structural gene sequences from the previouslydescribed human dendritic cell tropic SIN variant, SINDCchiron (ATCC#VR-2643, deposited Apr. 13, 1999). DNA clones used encompassing theentire genome of SINDCchiron virus (Gardner et al., ibid; WO 00/61772)were assembled using standard molecular biology techniques and methodswidely known to those of skill in the art (Rice et al., J. Virol.,61:3809-3819, 1987; and U.S. Pat. No. 6,015,694). The genomic SIN clonewas designated SINDCSP6gen.

Subsequently, the existing SIN structural proteins were replaced withthe hybrid capsid S129Vcapsid and VEE glycoproteins in the followingmanner. A fragment from tDH-S129V containing part of the hybrid capsidwas generated by PCR amplification with the following oligonucleotides:

S/VcVg1R (SEQ ID NO 54) atatatatggtcactagtgaccattgctcgcagttctccgScAatIIF (SEQ ID NO 55) gccgacagatcgttcgacgtc

The oligonucleotides were used 2 μM concentration with 0.1 μg of theappropriate template plasmid DNA in a 30 cycle PCR reaction, with Pfupolymerase as suggested by the supplier and with the addition of 10%DMSO. The general amplification protocol is illustrated below.

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 60 0.5 30 72 2

The PCR fragment was gel purified using QIAquick gel extraction kit.Another fragment containing the VEE glycoprotein fragment was obtainedfrom tDHVE2-120 by digestion with Spec and PmeI, and gel purification.The two fragments were mixed and ligated to an 11 kb fragment obtainedfrom the SINDCSP6gen clone by digestion with SpeI and PmeI, gelpurification, and treatment with shrimp alkaline phosphatase. Thepositive clones for the inserts were confirmed by sequencing and thisintermediate was called SrS129VcVg-interm. To restore the authentic3′-end in the genomic clone, the PsiI-PsiI fragment was regenerated byPCR with the following oligonucleotides

PsiIFd1N 5′ATATATATTTATAATTGGCTTGGTGCTGGCTACTATTGTGGCCATGTACGTG (SEQ.ID. NO. 53) CTGACCAACCAGAAACATAATTGACCGCTACGCCCCAATGATCC-3′ PsiR5′-GGCCGAAATCGGCAAAATCCC-3′ (SEQ. ID. NO. 54)at 2 μM concentration with 0.1 μg of the infectious clone plasmid DNA ina 30 cycle PCR reaction, with Vent polymerase as suggested by thesupplier and with the addition of 10% DMSO. The general amplificationprotocol illustrated below.

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 60 0.5 30 72 2The fragment was digested with PsiI, gel purified, and ligated toSrS129VcVg-interm that had also been digested with PsiI, gel purified,and treated with shrimp alkaline phosphate. The clones for the insertwere confirmed by sequencing and the final construct was designatedSrS129VcVg.

To construct a similar full-length cDNA clone containing the hybridS113V capsid, a fragment containing part of SIN sequences upstream ofthe capsid gene and the capsid gene encoding for aa1-113 was generatedusing the following oligonucleotides

-   Sic7082F-   5′-CACAGTTTTGAATGTTCGTTATCGC-3′ (SEQ. ID. NO. 55)-   S113R (see above)    at 2 μM concentration with 0.1 μg of the SINDCSP6gen construct in a    30 cycle PCR reaction, with Pfu polymerase as suggested by the    supplier and with the addition of 10% DMSO. The general    amplification protocol illustrated below.

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 60 0.5 30 72 2The fragment was gel purified using QIAquick gel extraction kit, and1/10^(th) of the reaction was mixed with fragment VEE(141-275) (seeabove, construction of all hybrid capsid genes). One PCR amplificationcycle was performed under the following conditions:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 42 1 1 72 3Then oligonucleotides Sic7082F and TRDCtR were added at 2 μMconcentration and the complete PCR amplification was performed asfollows:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 60 0.5 30 72 2

The PCR product was purified using the QIAquick kit, digested withBstZ17I and SapI, gel purified from agarose gel as described above, andligated to two fragments generated from plasmid SrS129VcVg that had alsobeen digested with BstZ17I and SapI to remove the existing capsid geneinsert. Clones containing the newly generated hybrid inserts wereverified by sequencing and the new construct was designated SIN113CVgly.

In order to generate virus, the SIN113CVgly construct was linearizedwith PmeI, transcribed in vitro using SP6 polymerase and the RNAtransfected into BHK cells. Progeny virus was harvested and passaged incells, with the infectious titer increasing to levels approaching 10⁹PFU/mL. A non-plaque purified stock of this chimeric SIN virus,designated SIN 113CVgly virus (deposited with ATCC May 31, 2001,PTA-3417), was then used as the source of RNA for cloning and sequencingby standard molecular biology techniques (e.g., those described above)to identify additional genetic determinants that provide this high levelof chimeric particle packaging. Individual genetic determinants arereadily incorporated back into the replicon and defective helperpackaging constructs of the present invention using teachings providedherein.

It is understood that the non-plaque-purified stock of chimeric SINvirus deposited with ATCC number may contain numerous genotypes andphenotypes not specifically disclosed herein that are considered part ofthe present invention. Persons having ordinary skill in the art couldeasily isolate individual phenotypes and or genotypes using plaquepurification techniques and sequence the isolated chimeric SIN usingprocedures known to those having ordinary skill in the art and disclosedherein.

Example 4 Generation of Alphavirus Replicon Particle Chimeras withHybrid Glycoproteins

In the case of a hybrid envelope glycoprotein using elements obtainedfrom both SIN and VEE, hybrid E2 glycoproteins were constructedcontaining the cytoplasmic tail (e.g., capsid binding portion) from SINand the transmembrane and ectodomain portions from VEE. Additionalconstructs with the opposite portions can also be derived. In someembodiments, it may also be desirable to include hybrids for both the E2and E1 glycoproteins, and to include hybrids that encompass thetransmembrane domain.

To demonstrate an increased efficiency of chimeric particle packagingusing such glycoprotein hybrids, a modified VEE-derived glycoprotein wasconstructed wherein the E2 tail was substituted with SIN-derived E2cytoplasmic tail. The fusion was done at the conserved cysteine residue(amino acid residue 390, both VEE and SIN E2) which is at the boundarybetween the transmembrane domain and the cytoplasmic tail (FIG. 5). Thechimera construct was generated by PCR amplification of two overlappingfragments one of which included part of VEE E2 glycoprotein sequenceupstream the cytoplasmic tail and part of the SIN E2 cytoplasmic tail.The second fragment included part of the SIN E2 cytoplasmic tail and VEE6K protein.

The first fragment was amplified from the construct VCR-DH-Vgly usingthe following oligonucleotides:

VE2F: 5′-atatatcaggggactccatcaccatgg-3′ (SEQ ID NO 35) (nts 7-27 arecomplementary to the VEE glycoprotein and include the NcoI site) VSGE2R:5′-gggattacggcgtttggggccagggcgtatggcgtcaggcactcacggcgcgcttt (SEQ ID NO36) gcaaaacagccaggtagacgc-3′ (nts 1-56 are SIN E2 cytoplasmic tailsequence, and nts. 57-77 are comple- mentary to VEE glycoproteinsequence)

The second fragment was amplified from the same plasmid using thefollowing primers:

VSGE3F: 5′gccccaaacgccgtaatcccaacttcgctggcactcttgtgctgcgttaggtcggccaatgctgagaccacctgggagtcctt(SEQ ID NO 37) g-3′ (nts.1-63 correspond to part of the SIN E2cytoplasmic tail sequence, and nts 64-84 are complementary to the VEEglycoproteins) VEE3′-1R:5′-ccaatcgccgcgagttctatgtaagcagcttgccaattgctgctgtatgc-3′ (SEQ ID NO 38)(complementary to VCR-DH Vgly downstream the glycoprotein open readingframe)

The oligonucleotides listed above were used at 2 μM concentration with0.1 μg of template plasmid DNA VCR-DH-Vgly in a 30 cycles PCR reactionwith Pfu Polymerase as suggested by the supplier, with the addition of10% DMSO. The amplification protocol is shown below.

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 60 0.5 30 72 2

The two amplified fragments were purified from agarose gel usingQIAquick gel extraction kit, and then an aliquot ( 1/10th) of eachfragment was used as templates for a second PCR amplification. The twofragments were mixed with Pfu Polymerase as suggested by supplier withthe addition of 10% DMSO. One PCR amplification cycle was performed:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 42 1 1 72 3

Then the VE2NtF and VEE3′-1R primers were added 2 μM concentration andthe PCR amplification was performed as follows:

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 0.5 60 0.5 30 72 2

The PCR product was purified using the QIAquick kit, digested with NcoIand NotI, gel purified from agarose gel as described above, and ligatedto plasmid tDH-Vgly that had also been digested with NcoI and NotI andpurified from agarose gel. Clones containing the inserts were verifiedby sequencing and the construct was designated tDH-VglySE2tail.

To demonstrate increased packaging of particles generated with such aglycoprotein chimera, plasmid DNA tDH-VglySE2tail was linearized withthe single restriction enzyme PmeI and RNA transcribed in vitro. The RNAwas co-transfected together with SINCR-GFP replicon RNA and thedefective helper RNA encoding SIN capsid protein. Transfected cells wereincubated at 34° C. for 24 hr, at which time the culture supernatant wascollected, clarified by centrifugation, serially diluted, and used toinfect naive BHK-21 cells for approximately 14 hr. Using flow cytometryanalysis the particles titers were determined and shown to be 2e³ IU/ml.This result showed that some low efficiency interaction is occurringbetween the glycoprotein chimera and SIN capsid.

To further increase the efficiency of chimeric particle packaging with ahybrid glycoprotein, additional constructs were generated. Alignment ofthe cytoplasmic tails from VEE and SIN (FIG. 5) shows the differences at10 residues, four of which are conservative changes. Interestingly, theresidues at positions 394 and 395 are charged in the SIN glycoprotein,while they are hydrophobic in VEE. Such difference might affect the E2functionality. Site directed mutagenesis using a PCR amplificationmethod was used to change the two residues in the constructtDH-VglySE2tail as follow:

Name Nucleotide change amino acid change Mutagenic oligos tDH-M1 A₂₁₅₁to C Glu₃₉₅ to Ala M1R (SEQ ID NO 39) 5′GTATGGCGTCA GGCACGCACGGCGCGCTTTG-3′ (SEQ ID NO 39) M1F 5′AGCGCGCCGT GCGTGCCTGACG CCATACGCC-3′(SEQ ID NO 40) tDH-M2 C₂₁₄₇ to G and G₂₁₄₈ Arg₃₉₄ to Val M2R (SEQ ID NO40) to T 5′ATGGCGTCAG GCACTCAACGCG CGCTTTGCAAAA C-3′ (SEQ ID NO 41) M2F5′TTTGCAAAGCG CGCGTTGAGTGC CTGACGCCATAC -3′ (SEQ ID NO 42) tDH-M3 A₂₁₅₁to C, C₂₁₄₇ to Arg₃₉₄-Glu₃₉₅ to Val-Ala M3R G, and G_(2148 to) T5′ATGGCGTCAG GCACGCAACGC GCGCTTTGCAAA AC-3′ (SEQ ID NO 43) M3F5′TTTGCAAAGCG CGCGTTGCGTGC CTGACGCCATAC -3′ (SEQ ID NO 44)

The mutagenized constructs were verified by sequencing. To quantitatepackaging by these new glycoprotein hybrids, the plasmid DNAs werelinearized with the single restriction enzyme PmeI and transcribed invitro. Each mutant RNA was then co-transfected together with theSINCR-GFP replicon RNA and defective helper RNA encoding SIN capsid.Transfected cells were incubated at 34° C. for 24 hr, at which time theculture supernatant was collected, clarified by centrifugation, seriallydiluted, and used to infect naive BK-21 cells for approximately 14 hrfor titer analysis. Using flow cytometry analysis, the particle titerswere determined and it was observed that the packaging efficiency wasincreased approximately 7-fold with M1.

Alternatively, and similarly to the capsid approach, it was possible tosubstitute the VEEglyco-E2 SIN tail chimera into a full-lengthalphavirus cDNA clone from which infectious virus may be obtained, anduse the chimeric virus genome to select naturally arising chimericparticle variants with further increased efficiency of packaging. Alarge plaque phenotype may be indicative of high titer virus. Thisinfectious chimera was constructed as follow. A fragment containingmostly SIN capsid sequence was generated by PCR in order to have a fewnucleotides added to its 3′ end corresponding to the VEE glycoproteinsequence and containing the SpeI restriction site. This fragment wasamplified from a human DC-tropic SIN infectious clone construct (Gardneret al., ibid) with the following primers:

ScAatIIF: (SEQ ID NO 45) 5′-gccgacagatcgttcgacgtc-3′ ScVg1R: (SEQ ID NO46) 5′-atatatatggtcactagtgaccactcttctgtcccttccg-3′These oligonucleotides were used at 2 μM concentration with 0.1 μg oftemplate plasmid DNA in a 30 cycles PCR reaction with Pfu Polymerase assuggested by the supplier with the addition of 10% DMSO. Theamplification protocol is shown below.

Temperature (° C.) Time (Min.) No. Cycles 94 2 1 94 0.5 60 0.5 30 72 2

The amplified fragment (450 bp) was cleaned using QIAquick PCRpurification kit, digested with AatII and SpeI, gel purified usingQIAquick gel extraction kit. A fragment (3.4 kb) containing the VEEglycoprotein-SIN E2tail and SIN 3′ UTR was generated by restrictiondigest from tDH-VE2tail using the enzymes SpeI-PmeI and gel purificationwith QIAquick gel extraction kit. This fragment and the PCR fragmentwere mixed and ligated together to plasmid DNA from the infectious clonethat had been also digested with AatII and PmeI, treated with Shrimpalkaline phosphatase, and gel purified. Positive clones for the insertwere verified by sequencing. Finally, to restore the authenticfull-length clone 3′-end, the PsiI-PsiI fragment was regenerated asdescribed for SrS129VcVg and the new construct was designated SrcVgSE2t.

SrcVgSE2t was linearized with the single restriction enzyme PmeI andtranscribed in vitro. The RNA was transfected into BHK cells.Transfected cells were incubated at 37° C. for 24 hr, at which time theculture supernatant was collected, clarified by centrifugation, and usedto infect naïve BHK-21 cells. Approximately 24 hr post-infection thesupernatant was collected, clarified by centrifugation, and used toinfect naïve BHK-21 cells again. At 24 hr post-infection a few viralplaques were observed, so the supernatant was collected, clarified andused for to infect two flasks of naïve BHK. The cells of one flask werecollected 16 hr post-infection and total RNA was extracted using Trizol(Gibco-BRL). The infection in the other flask was allowed to continuefor another 8 hrs and extensive cytopathic effects were observed in thecells indicating that large amounts of virus had been produced.

Total RNA extracted from the infected cells was used to amplify andclone capsid and glycoprotein sequences using RT-PCR. The reversetranscription was primed with either polydT or with the specific primer

-   VglyR: 5′-atatatatgcggccgctcaattatgtttctggttggtcag-3′ (SEQ ID NO 47)    The cDNA was then used for PCR amplification of the capsid sequence    with the primers SINNtF containing a XhoI site and SINCtR containing    a NotI site, and of the glycoprotein sequence with the primers VglyR    containing a NotI site and-   VglyF: 5′-atatatctcgagccgccagccatgtcactagtgaccac-3′ (SEQ ID NO 48)    containing a XhoI site. Both fragments were cleaned using QIAquick    PCR purification kit, digested with XhoI and NotI, gel purified    using QIAquick gel extraction kit and separately ligated to tDH that    had been previously digested with XhoI and NotI, gel purified and    treated with shrimp alkaline phosphatase. Ten clones for the capsid    fragment were sequenced to identify the possible adaptive    mutation(s). However, no mutations were found in the capsid region    indicating that either such mutations can only occur in the    glycoprotein sequences or that, since the RNA came from unpurified    plaques, the 10 clones did not completely represent the entire    adapted population.

Repeating the same analysis on RNA derived from 5 individual viralplaques still did not lead to identification of capsid adaptivemutations. The glycoprotein sequence from one plaque (P3) revealed thepresence of two amino acid changes at positions 380 (Val to Gly) and 391(Lys to Arg). Interestingly, the amino acid 380 is conserved betweenSindbis and at least three VEE strains (TRD, MAC10 and 6119) and aminoacid 391, which is the first residue in of the cytoplasmic tail, is aLys in the SIN glycoprotein sequences and MAC10 and 6119 but is a Arg inthe TRD strain. This might indicate that the location of these residuesplay a role in the correct conformation of the transmembrane-cytoplasmictail, which might stabilize the interactions between the glycoproteinsand the capsid, and may be further exploited as part of the presentinvention.

To test if this double mutation could increase packaging efficiency, a998 bp fragment (NcoI-MfeI) containing both mutations was swapped intotDH-VglySE2tail generating tDH-VglySE2tail-P3. Then, plasmid DNAtDH-VglySE2tail-P3 was linearized with the single restriction enzymePmeI and RNA transcribed in vitro. The RNA was co-transfected togetherwith SINCR-GFP replicon RNA and the defective helper RNA encoding SINcapsid protein. Transfected cells were incubated at 34° C. for 24 hr, atwhich time the culture supernatant was collected, clarified bycentrifugation, serially diluted, and used to infect naïve BHK-21 cellsfor approximately 14 hr. Using flow cytometry analysis, the particlestiters were determined and the efficiency of packaging increased 50 foldwith respect to VglySE2tail. Also, in the context of a hybrid VEEglycoprotein containing the SE2tail and the VEE E2-120 attenuatingmutation (VE2-120/SE2tail), the P3 mutations increased the packagingefficiency 200 fold.

Example 5 Generation of Alphavirus Replicon Particle Chimeras withHybrid Packaging Signal

To generate a highly efficient packaging system for a VEE repliconwithin Sindbis virus structural proteins, the well-defined RNA packagingsignal from SIN was inserted at various points within a VEE replicon.For this work the 132 nucleotide (nt.) core packaging signal from SINwas separately inserted into each of three different sites (FIG. 6)within the VEE-TRD replicon constructed in Example 1. Four chimericreplicons were generated. Chimera-1A and Chimera-1B were the names givento the constructs in which the SIN packaging signal was inserted at the3′ end of the VEE-TRD nsP4 gene, just prior to the nsP4 stop codon. TheChimera-2 replicon contains the SIN packaging signal in-frame, at theC-terminus of nsP3, substituting at the nucleotide level for a 102 bpsegment of nsP3. Finally, the Chimera-3 replicon resulted from theinsertion of the SIN packaging signal at the end of nsP3, just prior tothe nsP3 termination codon.

It is also contemplated by the inventors that the teachings herein mayprovide a unique opportunity to modify replicons and eukaryotic layeredvector initiation systems derived from any BSL-3 alphavirus (e.g., VEE),such that they may be treated as BSL-2 or BSL-1 constructs by reducingthe nucleotide sequence derived from the parental virus to less thantwo-thirds genome-length.

A) Chimera 1A, 1B:

A complicating factor for the construction of these chimeras lay in thefact that the subgenomic promoter of all alphaviruses overlaps the lastapproximately 100 nucleotides of nsP4. In order to place the SINpackaging signal at the end of nsP4 while maintaining a functionalsubgenomic promoter in the replicon vector for driving expression of theheterologous gene, it was necessary to alter the codon usage of the last80 nt. of nsP4 (upstream of the inserted SIN sequence) to eliminatetheir ability to bind the replicase complex. Simultaneously, the VEEsubgenomic promoter region was reconstituted downstream of the nsP4 stopcodon by duplicating the native sequence of a portion of the 3′ end ofnsP4 thought to be part of the subgenomic promoter recognition sequence.Chimera 1A and 1B differ by the length of reconstituted nsP4 sequencethat was added back to regenerate a functional subgenomic promoter: to−80 for CHIMERA-1A (FIG. 7), to −98 for CHIMERA-1B (FIG. 8).

Chimera 1A and 1B were prepared by cleaving pVCR-DH, an intermediateconstruct from the re-assembly phase of the pVCR construction described(above), with MscI and AscI. Into this vector was inserted either of twotripartite synthetic oligonucleotides coding, as described above, thelast 80 bp or so of nsP4 with non-native codon usage, followed by theSIN packaging signal (in frame) and nsP4 termination codon, followed bythe duplicated terminal 80 or 98 bp of native nsP4 sequence. Theoligonucleotides were designed to provide synthetic full duplex strandsthat were treated in the same manner as was described earlier for thereplicon synthesis. Sequence verified clones from this ligation weredigested with MscI and AscI, and the oligo fragment bearing the SINpackaging signal was substituted into the vector fragment of pVCR,digested similarly. The resulting final constructs for each was calledpVCR/CHIMERA-1A and pVCR/CHIMERA-1B. To evaluate the functionality ofthese constructs, the GFP gene was cloned into each using the uniqueBbvCI and NotI sites downstream of the subgenomic promoter and theconstructs were designated VCR-Chim1A-GFP and VCR-Chim1B-GFPrespectively.

B) Chimera 2:

Chimera-2 was prepared by cleavage of the VEE-replicon assemblyintermediate, pCMVkm2-(del XhoI/CelII)-VEE 9/10, from example 1, codingfor a portion of VEE nsP3 and nsP4 bounded by the MamI and BlnI sites ofthe replicon. XhoI cleavage of this vector deletes a 102 bp segment ofVEE nsP3. Into this cleaved vector was inserted a PCR product consistingof the SIN packaging signal flanked by terminal, in-frame, XhoI sites(FIG. 9). The template for this amplification was pSINCP and Pfu DNApolymerase was used with the following oligonucleotide primers.

5′Pr: 5′ -ATATCTCGAGAGGGATCACGGGAGAAAC-3′ (SEQ ID NO 49) 3′Pr:5′ -AGAGGAGCTCAAATACCACCGGCCCTAC-3′ (SEQ ID NO 50)

Resulting clones were validated for sequence and orientation. Onepositive clone was digested MamI -BlnI to generate a fragment used tosubstitute for the native MamI-BlnI segment of pVCR. The resultingplasmid was called pVCR/CHIMERA-2. The GFP gene was cloned into thisvector as described above for pVCR/CHIMERA-1A, -1B, generatingVCR-Chim2-GFP. It should be appreciated that the region of deletion innsP3 was selected based on convenient restriction endonuclease sites inthe plasmid DNA construct. Additional deletions that remove largerregions of nsP3 are also contemplated by the present invention and canbe performed readily by one of skill in the art.

C) Chimera-3:

Chimera-3 was prepared by modification of a replicon fragment fromexample 1, pCR2-9057b, which contained a portion of replicon fragments9+10, encoding the region of the junction of VEE nsP3 and nsP4.Insertion of the SIN packaging site was accomplished by overlapping PCRusing Pfu DNA polymerase and two sets of primers which amplified twoproducts across the junction in pCR2-9057b and which appended SINpackaging signal sequence tails to the resulting products. Similarly theSIN packaging signal was amplified from pSINCP with primers thatappended nsP3 and nsP4 sequence tails, respectively, at the 5′ and 3′ends of the product. See FIGS. 10 and 11 for detail of this strategyincluding primer sequences. The three PCR products were diluted, mixed,denatured, re-annealed, and extended with Pfu DNA polymerase to create achimeric overlap template for amplification utilizing the external nsP3and nsP4, 5′ and 3′ primers. This product was digested with XbaI andMluI and cloned into a similarly digested intermediate cloning vector,pCMVkm2 (zur Megede, J. Virol. 74:2628, 2000). To place the chimera inthe context of pVCR, the pCMVkm2/CHIMERA-3 intermediate was digestedwith MamI (5′) and SacI (3′) and co-ligated with a SacI-BlnI fragmentfrom pVCR (nt. 5620-6016 of pVCR) into the MamI/BlnI vector fragment ofpVCR. The resulting construct was called pVCR/CHIMERA-3. The GFP genewas cloned into this vector as described above for pVCR/CHIMERA-1A, -1B,generating VCR-Chim3-GFP.

To test the ability of these constructs to be packaged by Sindbisstructural proteins, the plasmids VCR-Chim1A-GFP, VCR-Chim1b-GFP,VCR-Chim2-GFP, and VCR-Chim3-GFP were linearized with the singlerestriction enzyme PmeI and RNA transcribed in vitro. The RNA wasco-transfected together with defective helper RNAs encoding SIN capsidand glycoproteins from constructs VCR-DH-Sglydl160 and VCR-DH-Scap alsolinearized with PmeI. Transfected cells were incubated at 34° C. for 24hr, at which time the culture supernatants were collected, clarified bycentrifugation, serially diluted, and used to infect naive BHK-21 cellsfor approximately 14 hr. Using flow cytometry analysis the particletiters were determined. The results below showed that three chimerascould be packaged efficiently by the SIN structural proteins. Chimera 1Awas not expressing GFP and it was not determined whether this was due toa defect in the subgenomic transcription or in the RNA replication.

Replicon Structural proteins Titers VCR-Chimera1A SIN 0 VCR-Chimera1BSIN 3.8E⁷ Iu/ml VCR-Chimera 2 SIN 9.6E⁷ Iu/ml VCR-Chimera1A SIN   3E⁷Iu/mlConstruction of Chimera 2.1

To further reduce the amount of parental VEE virus sequence present inthe pVCR-Chimera2 replicon, the 3′ NTR (also known as 3′ sequencerequired for nonstructural protein-mediated amplification, or 3′ UTR)sequence from VEE was removed in its entirety and replaced by the 3′ NTRfrom SIN. Plasmid SINCR-GFP (Garner et al., 2000 ibid.) was digestedwith NotI and PmeI, the 466 bp fragment was gel purified using QIAquickgel extraction kit and ligated to both pVCR-Chimera2 and VCR-Chim2-GFPthat had been previously digested with NotI and PmeI, gel purified andtreated with shrimp alkaline phosphatase. Positive clones were verifiedand the constructs designated VCR-Chim2.1 and VCR-Chim2.1-GFP. Theseconstructs now differ from the parental VEE virus genome by the deletionof multiple VEE sequences (e.g., region of nsP3, structural proteingenes, 3′ NTR).

To test the functionality of the new chimera replicon vectorconfiguration, plasmid VCR-Chim2.1-GFP was linearized with the singlerestriction enzyme PmeI and RNA transcribed in vitro. The RNA wasco-transfected together with defective helper RNAs encoding SIN capsidand glycoproteins from constructs VCR-DH-Sglydl160 and VCR-DH-Scap alsolinearized with PmeI. Transfected cells were incubated at 34° C. for 24hr, at which time the culture supernatants were collected, clarified bycentrifugation, serially diluted, and used to infect naive BHK-21 cellsfor approximately 14 hr. Using flow cytometry analysis, the particletiters were determined to be the same titers as VCR-Chim2-GFP,demonstrating that deletion of the native 3′ NTR and replacement with aheterologous alphavirus 3′ NTR (e.g., SIN 3′ NTR) maintainsfunctionality in the VEE replicon.

Alternatively, as a means to reduce the overall VEE-derived sequences inVCR-Chimera2, the 3 ′NTR was reduced to a minimal sequence containingthe 19nt conserved CSE. Such a modified 3′NTR was generated usingoverlapping oligonucleotides:

Vred2F 5′-ggccgcttttcttttccgaatcggattttgtttttaat-3′ Vred2R5′-attaaaaacaaaatccgattcggaaaagaaaagc-3′ VEE3F see VCR-DH constructionfor oligonucleo- tide sequences VEE3R VEE4F VEE4R

Each pair of forward and reverse oligonucleotides (e.g., Vred2F withVred2R, VEE2F with VEE2R, etc.) were mixed, phosphorylated, denatured,and slowly annealed. Then the 3 pairs of annealed oligonucleotides weremixed together, ligated to each other, digested with enzymes NotI andPmeI, gel purified using a QIAquick gel extraction kit, and ligated tothe VCR-Chim2-GFP that had been previously digested with the sameenzymes to delete the full length 3′NTR, gel purified and treated withshrimp alkaline phosphatase. Positive clones for the fragment wereverified by sequencing. This construct was called VCR-Chim2.2-GFP.

To confirm functionality of this chimera replicon vector configuration,plasmid VCR-Chim2.2-GFP was linearized with the single restrictionenzyme PmeI and RNA transcribed in vitro. The RNA was co-transfectedtogether with defective helper RNAs encoding SIN capsid andglycoproteins from constructs VCR-DH-Sglydl160 and VCR-DH-Scap alsolinearized with PmeI. Transfected cells were incubated at 34° C. for 24hr, at which time the culture supernatants were collected, clarified bycentrifugation, serially diluted, and used to infect naïve BHK-21 cellsfor approximately 14 hr. Using flow cytometry analysis, the particletiters were determined to be the similar to VCR-Chim2-GFP, demonstratingthat reducing the size of the 3′NTR from 117bp to 37 bp and replacementmaintains functionality of the replicon.

Similar to the above replicon vectors for use as RNA or repliconparticles, alphavirus DNA-based replicons that function directly withina eukaryotic cell (e.g., Eukaryotic Layered Vector Initiation Systems)may be derived by one of skill in the art, using the teachings providedherein. Such DNA-based replicons may be deleted of a variety of parentalvirus sequences for example, including, but not limited to, sequencesfrom the nsP3 carboxy terminal region, structural protein gene region,3° CSE region, and the like.

Example 6 Use of Different Structural Proteins for Delivery of RepliconRNA

An HIV antigen was expressed from SIN replicon RNA packaged with eitherSIN or VEE structural proteins, and from VEE replicon RNA packaged witheither SIN or VEE structural proteins as follows. Specifically, afragment containing the heterologous gene sequence encodingcodon-optimized HIV p55gag (zur Megede, J. Virol. 74:2628, 2000) fromplasmid pCMVKm2.GagMod.SF2 was inserted into the SINCR replicon vector(Gardner et al., 2000, ibid) at the XhoI-NotI sites, into the VCRreplicon vector at the BbvCI-NotI sites and into the VCR-Chim2.1 vectorat the BbvCI-MfeI sites. The p55gag encoding replicon constructs weredesignated SINCR-p55gag, VCR-p55gag, and VCR-Chim2.1-p55gag,respectively. To produce SIN, VEE and chimera replicon particlesexpressing p55gag, the above plasmids were linearized with the singlerestriction enzyme PmeI and RNA transcribed was in vitro. The RNA wasco-transfected together with defective helper RNA encoding for theappropriate structural proteins which were transcribed from the PmeIlinearized plasmids as shown below:

Particles Replicon Caspid Glycoproteins SIN SINCR-p55gag SINdl-cap (Poloet al, tDH-VUTR-Sglydl160 1999, ibid) VEE VCR-p55gag VCR-DH-VcapVCR-DH-VE2-120 SINrep/VEEenv SINCR-p55gag tDH-S113VcaptDH-VUTR-Sglydl160 VEErep/SINenv VCR-Chim2.1p55gag VCR-DH-ScapVCR-DH-Vglydl160

Transfected cells were incubated at 34° C., supernatants collected at 20hr and 36 hr, followed by clarification by centrifugation, andchromatographic purification as described previously (PCT WO 01/92552).

Particle titers were determined by intracellular staining for gagexpression in BHK21 cells infected for 16 hrs with serial dilution ofpurified particle preparations. The cells were first permeabilized andfixed with Cytofix/Cytoperm Kit (Pharmingen), then stained forintracellular p55gag with FITC conjugated antibodies to HIV-1 coreantigen (Coulter). Using flow cytometry analysis, the percentage of gagpositive cells were determined and used to calculate the particletiters.

Immunogenicity in rodent models was determined after immunization withthe different alphavirus replicon particle preparations expressing HIVp55gag, at doses of 10⁶ or 10⁷ IU replicon particle doses (FIG. 12).Each was found to be immunogenic and one chimera, VEErep/SINenv, wasfound to be a particularly potent immunogen.

Demonstration of sequential immunization of rodents or primates withalphavirus replicon particles, such as the above replicon particles,differing in their structural proteins, may be performed using a varietyof routes (e.g., intramuscular, intradermal, subcutaneous, intranasal)and with dosages ranging from 10³ IU up to 10⁸ IU, or greater. Forexample, primates are immunized first with 10⁷ SINCR-p55gag particlescontaining VEE structural proteins in 0.5 mL of PBS diluent, by asubcutaneous route. The same materials are then administered a secondtime 30 days later, by the same route of injection. Approximately 6-12months later, the animals are then immunized one or more times with 10⁷SINCR-p55gag particles containing SIN structural proteins in 0.5 mL ofPBS diluent, by an intramuscular route. Demonstration of immunogenicityis performed using standard assays and may be compared to parallelanimals that received only a single type of replicon particle at time ofadministration.

The preceding examples have described various techniques suitable forpreparing chimeric alphavirus particles using nucleic acids,nonstructural proteins and structural proteins, as well as portionsthereof, derived from two different alphaviruses. However, one ofordinary skill in the art, using the teaching provided herein, couldprepare chimeric alphavirus particles from three or more viruses withoutundue experimentation. In would be logical to combine the teachingsfound herein with the teachings of other relevant technical disclosuresgenerally available to those skilled in the art including, but notlimited to, patents, patent applications, scientific journals,scientific treatise and standard references and textbooks.

For example, alphavirus chimeric particles are made using SIN repliconvectors and at least two defective helper RNA molecules. The repliconRNA encodes for SIN non-structural proteins, a VEE packaging signal anda heterologous gene of interest. The first defective helper RNA encodesfor a hybrid capsid protein having a VEE RNA binding domain and a WEEglycoprotein interaction domain. The second defective helper RNA encodesfor WEE glycoprotein. The resulting chimeric alphavirus particles havenucleic acid derived from SIN with a VEE/WEB hybrid capsid and a WEEenvelope glycoprotein.

In another example, a chimeric alphavirus particle is made in accordancewith the teachings of the present invention where a SIN replicon havingSIN non-structural proteins and a heterologous gene of interest iscombined with two defective helper RNA molecules. The first defectivehelper RNA encodes for a hybrid capsid having a SIN RNA binding domainand a SFV glycoprotein interaction domain. The second defective helperRNA encodes for a hybrid glycoprotein having a SFV cytoplasmic tail withthe remainder of the glycoprotein envelope provided by VEE. Theresulting chimeric alphavirus particle has SIN nucleic acids with aheterologous gene of interest encapsidated in a SIN/SFV hybrid capsidwith a SFV/VEE hybrid envelope glycoprotein, the outer ectodomainportion of the glycoprotein being derived from VEE.

In yet another example four different alphaviruses are used to preparethe chimeric alphavirus particle. In this example a SIN replicon RNAencoding for SIN non-structural proteins, a VEE packaging signal and aheterologous gene of interest is provided. A first defective helper RNAencodes for a hybrid capsid having a VEE RNA binding domain and a WEEglycoprotein interaction domain. The second defective helper RNA encodesfor a hybrid glycoprotein having a WEE cytoplasmic tail with theremainder of the glycoprotein being provided by SFV. The resultingchimeric alphavirus particle has SIN RNA and a heterologous gene ofinterest, a VEE/WEE hybrid capsid and a WEE/SFV hybrid glycoprotein, theouter ectodomain portion of the glycoprotein being derived from SFV.

Many other combinations are possible and the preceding examples serve toillustrate the present invention's tremendous versatility. Therefore,these non-limiting examples represent only a few of the numerouschimeric alphavirus particles that can be made in accordance with theteachings of the present invention.

Example 7 Use of Alphavirus Replicon Vectors and Defective Helpers withDifferent Control Elements

To produce alphavirus replicon particles using vector (e.g., repliconRNA, eukaryotic layered vector initiation system) and packaging (e.g.,defective helper, structural protein expression cassette) componentswith different control elements, a wide variety of combinations may beutilized according to the present invention. For example, a SIN plasmidDNA-based replicon (eukaryotic layered vector initiation system) can beconstructed to contain a different 3′ sequence required fornonstructural protein-mediated amplification (3° CSE) than contained inthe structural protein expression cassettes of a SIN packaging cellline. More specifically, modification of the SIN 3′ end to incorporate apolyadenylation signal derived from the bovine growth hormone gene isperformed as described below. The resulting sequence:

(SEQ ID NO 56) GCGGCCGCCGCTACGCCCCAATGATCCGACCAGCAAAACTCGATGTACTTCCGAGGAACTGATGTGCATAATGCATCAGGCTGGTACATTAGATCCCCGCTTACCGCGGGCAATATAGCAACACTAAAAACTCGATGTACTTCCGAGGAAGCGCAGTGCATAATGCTGCGCAGTGTTGCCACATAACCACTATATTAACCATTTATCTAGCGGACGCCAAAAACTCAATGTATTTCTGAGGAAGCGTGGTGCATAATGCCACGCAGCGTCTGCATAACTTTTATTATTTCTTTTATTAATCAAATAAATTTTGTTTTTAACATTTCAAAAAAAAAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGGTTTAAACthus is engineered into the SIN plasmid construct. This new sequence issubstituted for the existing 3′-end, synthetic polyA-tract, ribozyme,and BHGpolyA site of plasmid pSINCP (See, WO 01/81690) as follows.Plasmid pSINCP-bgal (pSINCP expressing bgal) is deleted of theaforementioned elements by PCR with the following primers: NPSfwd:

(SEQ ID NO 57) 5′ACAGACAGACCGCGGCCGCACAGACAGACGTTTAAACGTGGGCGAAGAACTCCAGCATGAGATCCwhich contains a NotI site (12-19 nts.), a PmeI site (30-37 nt), and38-65 nts that are complementary to SINCP-bgal sequences downstream ofthe aforementioned elements, a NotI site precedes them.

-   NPSrev:-   5′-TTCGCCAGGCTCAAGGCGCGCATGCCCGAC (SEQ ID NO 58)    which is complementary to the plasmid backbone region containing the    SphI site. The amplified 492 bp fragment is purified from agarose    gel using QIAquick gel extraction kit, digested with NotI and SphI    and ligated to SINCP-bgal that has also been digested with NotI and    SphI to remove the existing sequence (1106 bp). Clones containing    the newly generated fragment are verified by sequencing and the    intermediate construct is called SINCPt-bgal. The new 3′ end is then    generated using overlapping oligonucleotides:

SINpA1F5′-tcgacccgggcggccgccgctacgccccaatgatccgaccagcaaaactcgatgtacttccgaggaactg-3′(SEQ ID NO 59) SINpA1R 5′-ggtcggatcattggggcgtagcggcggccgcccgggtcga-3′(SEQ ID NO 60) SINpA2F5′-atgtgcataatgcatcaggctggtacattagatccccgcttaccgcgggcaatatagcaacactaaaaac-3′(SEQ ID NO 61) SINpA2R5′-agcggggatctaatgtaccagcctgatgcattatgcacatcagttcctcggaagtacatcgagttttgct-3′(SEQ ID NO 62) SINpA3F5′-tcgatgtacttccgaggaagcgcagtgcataatgctgcgcagtgttgccacataaccactatattaacca-3′(SEQ ID NO 63) SINpA3R5′-gcgcagcattatgcactgcgcttcctcggaagtacatcgagtttttagtgttgctatattgcccgcggta-3′(SEQ ID NO 64) SINpA4F5′-tttatctagcggacgccaaaaactcaatgtatttctgaggaagcgtggtgcataatgccacgcagcgtct-3′(SEQ ID NO 65) SINpA4R5′-cctcagaaatacattgagtttttggcgtccgctagataaatggttaatatagtggttatgtggcaacact-3′(SEQ ID NO 66) SINpA5F5′-gcataacttttattatttcttttattaatcaaataaattttgtttttaacatttcaaaaaaaaagtaggtg-3′(SEQ ID NO 67) SINpA5R5′-aacaaaatttatttgattaataaaagaaataataaaagttatgcagacgctgcgtggcattatgcaccacgctt-3′(SEQ ID NO 68) SINpA6F5′-tcattctattctggggggtggggtgggggtttaaacatcatgatcg-3′ (SEQ ID NO 69)SINpA6R5′-cgatcatgatgtttaaacccccaccccaccccccagaatagaatgacacctactttttttttgaaatgttaaa-(SEQ ID NO 70) 3′The oligonucleotides are mixed, phosphorylated, denatured, slowlyannealed, and ligated. After inactivating the ligase, the DNA isdigested with the enzymes NotI and PmeI, gel purified using the QIAquickgel extraction kit and ligated to SINCPt-bgal digested with the sameenzymes and treated with alkaline phosphatase. Clones containing thenewly generated fragment are verified by sequencing and the finalconstruct is called SINCP-pA-bgal.

To produce replicon particles this plasmid is transfected into a SINpackaging cell line that contains structural protein expressioncassettes, which do not have similarly modified 3′-end sequences (Poloet al., 1999. Proc. Natl. Acad. Sci. USA, 96:4598-603). Afterappropriate incubation, the replicon particles are harvested andpurified as describe above.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A chimeric alphavirus particle, comprising: RNA derived from one ormore alphaviruses; and a capsid protein, wherein said capsid protein isderived from two or more different alphaviruses.
 2. A chimericalphavirus particle comprising RNA derived from a first alphavirus andstructural proteins, wherein said structural proteins comprise: (a) ahybrid capsid protein having (i) an RNA binding domain derived from saidfirst alphavirus and (ii) an envelope glycoprotein interaction domainderived from a second alphavirus which is different from the firstalphavirus; and (b) an envelope glycoprotein derived from said secondalphavirus.
 3. The chimeric alphavirus particle of claim 1, comprising:RNA derived from a first alphavirus; and an envelope glycoprotein having(i) a cytoplasmic tail portion derived from said first alphavirus and(ii) at least 95% of the remaining portion derived from a secondalphavirus, wherein the second alphavirus is different from the firstalphavirus.
 4. A chimeric alphavirus particle comprising (1) RNAencoding one or more nonstructural proteins derived from a firstalphavirus, (2) a packaging signal derived from a second alphavirusdifferent from said first alphavirus, wherein said packaging signal isinserted into a site selected from the group consisting of the junctionof nsP3 with nsP4 and a deletion in a nonstructural protein gene; (3) acapsid protein derived from said second alphavirus; and (4) an envelopeprotein derived from an alphavirus different from said first alphavirus.5. The chimeric alphavirus particle of claim 4, wherein packaging signalis inserted into a carboxy terminal deletion in a nonstructural proteingene.
 6. The chimeric alphavirus particle of claim 4, wherein saidenvelope protein is derived from said second alphavirus.
 7. Thealphavirus particle of claim 1, wherein said particle is a repliconparticle and said RNA comprises, in 5′ to 3′ order (i) a 5′ sequencerequired for nonstructural protein-mediated amplification, (ii) anucleotide sequence encoding alphavirus nonstructural proteins, (iii) ameans for expressing a heterologous nucleic acid, (iv) the heterologousnucleic acid sequence, said heterologous nucleic acid sequence replacingan alphavirus structural protein gene, (v) a 3′ sequence required fornonstructural protein-mediated amplification, and (vi) a polyadenylatetract.
 8. The alphavirus particle of claim 2, wherein said particle is areplicon particle and said RNA comprises, in 5′ to 3′ order (i) a 5′sequence required for nonstructural protein-mediated amplification, (ii)a nucleotide sequence encoding alphavirus nonstructural proteins, (iii)a means for expressing a heterologous nucleic acid, (iv) theheterologous nucleic acid sequence, said heterologous nucleic acidsequence replacing an alphavirus structural protein gene, (v) a 3′sequence required for nonstructural protein-mediated amplification, and(vi) a polyadenylate tract.
 9. An alphavirus particle of claim 3,wherein said particle is a replicon particle and said RNA comprises, in5′ to 3′ order (i) a 5′ sequence required for nonstructuralprotein-mediated amplification, (ii) a nucleotide sequence encodingalphavirus nonstructural proteins, (iii) a means for expressing aheterologous nucleic acid, (iv) the heterologous nucleic acid sequence,said heterologous nucleic acid sequence replacing an alphavirusstructural protein gene, (v) a 3′ sequence required for nonstructuralprotein-mediated amplification, and (vi) a polyadenylate tract.
 10. Analphavirus particle of claim 4, wherein said particle is a repliconparticle and said RNA comprises, in 5′ to 3′ order (i) a 5′ sequencerequired for nonstructural protein-mediated amplification, (ii) anucleotide sequence encoding alphavirus nonstructural proteins, (iii) ameans for expressing a heterologous nucleic acid, (iv) the heterologousnucleic acid sequence, said heterologous nucleic acid sequence replacingan alphavirus structural protein gene, (v) a 3′ sequence required fornonstructural protein-mediated amplification, and (vi) a polyadenylatetract.
 11. The chimeric alphavirus particle of claim 1, wherein one ofsaid two or more alphaviruses is Sindbis virus (SIN) and wherein anotherof said two or more alphaviruses is Venezuelan equine encephalitis virus(VEE).
 12. The chimeric alphavirus particle of claim 1, wherein one ofsaid one or more alphaviruses is VEE and wherein one of said two or morealphaviruses is SIN.
 13. The chimeric alphavirus particle of claim 2,wherein said first alphavirus is SIN and wherein said second alphavirusis VEE.
 14. The chimeric alphavirus particle of claim 2, wherein saidfirst alphavirus is VEE and wherein said second alphavirus is SIN. 15.The chimeric alphavirus particle of claim 3, wherein said firstalphavirus is SIN and wherein said second alphavirus is VEE.
 16. Thechimeric alphavirus particle of claim 3, wherein said first alphavinisis VEE and wherein said second alphavirus is SIN.
 17. The chimericalphavirus particle of claim 4, wherein said first alphavinis is SIN andwherein said second alphavirus is VEE.
 18. The chimeric alphavirusparticle of claim 4, wherein said first alphavinis is VEE and whereinsaid second alphavirus is SIN.
 19. The chimeric alphavirus particle ofclaim 7, wherein one of said two or more alphaviruses is SIN and whereinanother of said two or more alphaviruses is VEE.
 20. The chimericalphavirus particle of claim 7, wherein one of said two or morealphaviruses is VEE and wherein one of said two or more alphaviruses isSIN.
 21. The chimeric alphavirus particle of claim 8, wherein said firstalphavirus is SIN and wherein said second alphavirus is VEE.
 22. Thechimeric alphavirus particle of claim 8, wherein said first alphavirusis VEE and wherein said second alphavirus is SIN.
 23. The chimericalphavirus particle of claim 9, wherein said first alphavirus is SIN andwherein said second alphavirus is VEE.
 24. The chimeric alphavirusparticle of claim 9, wherein said first alphavirus is VEE and whereinsaid second alphavirus is SIN.
 25. The chimeric alphavirus particle ofclaim 10, wherein said first alphavirus is SIN and wherein said secondalphavirus is VEE.
 26. The chimeric alphavirus particle of claim 10,wherein said first alphavirus is VEE and wherein said second alphavirusis SIN.
 27. The alphavirus particle of claim 1, wherein said RNA furthercomprises a heterologous nucleic acid sequence.
 28. The alphavirusparticle of claim 27, wherein said heterologous nucleic acid replaces atleast one alphavirus structural protein.
 29. The alphavirus repliconparticle of claim 27, wherein heterologous nucleic acid sequence encodesfor a therapeutic agent or an immunogen.
 30. A method for producingalphavirus replicon particles, comprising introducing into a host cell:a) an alphavirus replicon RNA derived from one or more alphaviruses,wherein said replicon RNA further comprises one or more heterologoussequence(s); and b) at least one separate defective helper RNA(s)encoding structural protein(s) absent from the replicon RNA, wherein oneof said structural proteins is a capsid protein derived from two or moredifferent alphaviruses, wherein alphavirus replicon particles areproduced.
 31. The method of claim 30, wherein said replicon RNA isderived from a first alphavirus and wherein said structural proteinscomprise (a) a hybrid capsid protein having (i) an RNA binding domainderived from said first alphavirus and (ii) an envelope glycoproteininteraction domain derived from a second alphavirus; and (b) an envelopeglycoprotein derived from said second alphavirus.
 32. A method forproducing alphavirus replicon particles, comprising introducing into ahost cell: a) an alphavirus replicon RNA encoding one or morenonstructural proteins from a first alphavirus, a packaging signalderived from a second alphavirus which is different from the firstalphavirus, and one or more heterologous sequence(s), wherein saidpackaging signal is inserted into a site selected from the groupconsisting of the junction of nsP3 with nsP4 and a deletion in anonstructural protein gene; and b) at least one separate defectivehelper RNA(s) encoding structural protein(s) absent from the repliconRNA, wherein at least one of said structural proteins is a capsidprotein derived from said second alphavirus, and at least one of saidstructural proteins is an envelope protein derived from an alphavirusdifferent from said first alphavirus, wherein alphavirus repliconparticles are produced.
 33. A method of generating an immune response ina mammal, the method comprising administering a chimeric alphavirusparticle of claim 29 to said mammal, thereby generating an immuneresponse.
 34. The chimeric alphavirus particle of claim 4, wherein saidRNA further comprises a heterologous nucleic acid sequence.
 35. Thechimeric alphavirus particle of claim 34, wherein said heterologousnucleic acid sequences replaces at least one alphavirus structuralprotein.
 36. The chimeric alphavirus particle of claim 34, wherein saidheterologous nucleic acid sequence encodes a therapeutic agent or animmunogen.
 37. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 7. 38. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 8. 39. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 9. 40. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 10. 41. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 19. 42. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 20. 43. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 21. 44. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 22. 45. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 23. 46. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 24. 47. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 25. 48. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 26. 49. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 27. 50. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 28. 51. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 29. 52. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 34. 53. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 35. 54. A method of generating an immune response in a mammalcomprising administering to said mammal a chimeric alphavirus particleof claim
 36. 55. The method of claim 30 wherein the replicon RNA isderived from a first alphavirus and wherein said structural proteinscomprise an envelope glycoprotein having (i) a cytoplasmic tail portionderived from the first alphavirus and (ii) at least 95% of the remainingportion derived from a second alphavirus, wherein the second alphavirusis different from the first alphavirus.